Bachelor-thesis Antinéa BABARIT

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ER-Import Defects in Phosphorylation Mutants of Sbh1p

BACHELOR THESIS (B.SC)

Submitted by Antinéa BABARIT

Human and Molecular Biology Center - Zentrum für Human- und Molekularbiologie (ZHMB)

Faculty of Natural Sciences and Technology III and Faculty of Medicine

Saarland University, Germany

Saarbrücken, July 2015

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The experimental work depicted in this thesis has been carried out at the department of Microbiology at Saarland University, Germany.

First Reviewer: Prof. Dr. Karin Römisch

Second Reviewer: Joseph Schacherer

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Biology is the study of complicated things that have the appearance of having been designed with a purpose.

- Richard Dawkins

Success consists of going from failure to failure without loss of enthusiasm.

- Winston Churchill

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Table of contents Table of contents ............................................................................................................................4

List of Figures ................................................................................................................................. 7

List of Tables ...................................................................................................................................8

I. Introduction ............................................................................................................................... 9

1. From translation to secretion: The Secretory Pathway ...................................................... 9

1.1 - Targeting to the ER ...........................................................................................................9

1.2 - ER to Golgi trafficking .................................................................................................... 10

1.3 - Secretion and Exocyst ...................................................................................................... 11

2. Across the membrane: Protein Translocation into the ER .............................................. 12

2.1 - The Sec61 complex ........................................................................................................... 12

2.1.1 - The Sec61p Subunit ................................................................................................. 13

2.1.2 - The Sbh1p Subunit .................................................................................................. 13

2.1.3 - The Sss1p Subunit .................................................................................................. 14

2.2 - The Ssh1 Complex ........................................................................................................... 14

2.3 - The Sec Complex ............................................................................................................. 15

3. Post-translational modifications: Processing in the ER....................................................15

3.1 - Evolving into glycoprotein............................................................................................... 15

3.1.1 - N-linked glycosylation ............................................................................................15

3.1.2 - Oligosaccharyl Transferase Complex .................................................................. 16

3.2 - More post-translational events ..................................................................................... 16

3.3 - Protein inspection - ER Quality Control .......................................................................17

3.4 - The good, the bad and the misfolded - ER-Associated Degradation ...........................17

3.5 - Stress in the ER and Unfolded Protein Response ......................................................... 19

4. Aim of this study ................................................................................................................... 20

II. Materials and Methods ........................................................................................................... 22

1. Materials ................................................................................................................................. 22

1.1 - Laboratory equipment, chemicals, reagents and consumables ................................... 22

1.2 - Media and Buffers ........................................................................................................... 28

1.3 - Bacterial and yeast strains ............................................................................................. 33

1.4 - Plasmids .......................................................................................................................... 35

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1.5 - Antibodies ........................................................................................................................ 36

1.6 - Softwares ......................................................................................................................... 37

2. Methods ................................................................................................................................. 37

2.1 - Sterilization ..................................................................................................................... 37

2.2 - Growth of S. cerevisiae ................................................................................................... 37

2.3 - Growth of E. coli ............................................................................................................. 37

2.4 - Plasmid Extraction......................................................................................................... 37

2.5 - Transformation of S. cerevisiae ..................................................................................... 38

2.6 - Preparation of Cell Extracts .......................................................................................... 38

2.7 - Protein Gel Electrophoresis and Western Blot Analysis ............................................. 38

2.7.1 - Protein Gel Electrophoresis .................................................................................. 38

2.7.2 - Western Blot Analysis .......................................................................................... 39

2.7.3 - Membrane stripping ............................................................................................. 39

2.8 - Cycloheximide Chase Experiments ............................................................................... 39

2.9 - Pulse Experiments .......................................................................................................... 39

2.9.1 - Pulse Labeling ........................................................................................................ 39

2.9.2 - Immunoprecipitation .......................................................................................... 40

2.9.3 - Gel Electrophoresis and Revealing .................................................................... 40

2.10 - Native Immunoprecipitation ...................................................................................... 40

III. Results ..................................................................................................................................... 42

1. Import of Glucosidase I into the ER .................................................................................... 42

1.1 - Phosphorylation- and double transmembrane mutants .............................................. 42

1.2 - Combined mutants .........................................................................................................44

2. Oligosaccharyl Transferase Complex and Sbh1p .............................................................. 46

3. ER-Associated Degradation Defects ................................................................................... 47

3.1 - Phosphorylation- and double transmembrane mutants ............................................. 47

3.2 - Combined mutants ........................................................................................................ 49

4. Test on Antibodies Directed Against Sbh1p ..................................................................... 49

IV. Discussion ............................................................................................................................... 52

1. Import of Glucosidase I into the ER .................................................................................... 52

2. Oligosaccharyl Transferase Complex and Sbh1 ................................................................. 53

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3. ER-Associated Degradation Defects ................................................................................... 54

4. Test on Antibodies Directed Against Sbh1p ...................................................................... 56

V. Conclusion ................................................................................................................................ 57

VI. References ............................................................................................................................... 58

VII. Abbreviations ........................................................................................................................62

VIII. Annex .................................................................................................................................... 65

1. Auxotrophic amino-acids ..................................................................................................... 65

2. Supplementary figures......................................................................................................... 66

IX. Acknowledgements ............................................................................................................... 68

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List of Figures

Figure 1 - The SRP targeting cycle. ............................................................................................. 10

Figure 2 - ER to Golgi trafficking. ................................................................................................ 11

Figure 3 - Sec61 complex. .............................................................................................................. 13

Figure 4 - Calnexin/Calreticulin cycle. ...................................................................................... 18

Figure 5 - ERAD Pathway. ........................................................................................................... 20

Figure 6 - Import of Gls1 in SBH1 mutant strains. .................................................................... 43

Figure 7 - Amount of Gls1 in the cell for different SBH1 mutants. .......................................... 43

Figure 8 - Import of Gls1 in SBH1 mutant strains. ................................................................... 44

Figure 9 - Import of Gls1 in SBH1 combined mutant strains. .................................................. 45

Figure 10 - Amount of Gls1 in the cell for different combined SBH1 mutants. ...................... 45

Figure 11 - Import of CPY in SBH1 mutants. ............................................................................. 46

Figure 12 - Degradation of CPY* in SBH1 mutant strains over time. .................................... 48

Figure 13 - Degradation of CPY* in combined SBH1 mutant strains over time. .................... 50

Figure 14 -Testing of a new Sbh1 antibody and comparison with the previously used one ..51

Figure 15 - Degradation of CPY* in SBH1 mutant strains over time. ..................................... 66

Figure 16 - Import of CPY in SBH1 mutant strains. .................................................................. 67

Figure 17 - Import of CPY in SBH1 combined mutant strains. ................................................ 67

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List of Tables

Table 1 - Chemicals and reagents used in this study ................................................................ 22

Table 2 - Laboratory equipment used in this study ................................................................. 25

Table 3 - Consumables used in this study ................................................................................. 27

Table 4 - Composition of the media used in this study ........................................................... 28

Table 5 - Composition of the solutions for yeast transformation ........................................... 29

Table 6 - Composition of the solutions for cell extract preparation ...................................... 30

Table 7 - Composition of the solutions for SDS-PAGE and Western Blot ............................. 30

Table 8 - Composition of the solutions for pulse experiments ................................................ 31

Table 9 - Composition of the solutions for native immunoprecipitation.............................. 33

Table 10 - Escherichia coli (E.coli) strains used in this study .................................................. 33

Table 11 - S. cerevisiae strains used in this study ...................................................................... 34

Table 12 - Plasmids used in this study ........................................................................................ 35

Table 13 - Antibodies used in this study .................................................................................... 36

Table 14 - Softwares used in this study ...................................................................................... 37

Table 15 - Composition of Synthetic Complete amino acid drop-out mixture* for S. cerevisiae ....................................................................................................................................... 65

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I. Introduction

1. FROM TRANSLATION TO SECRETION: THE SECRETORY PATHWAY

Proteins are the basis element of all living cells. They have many roles in the cell life, such as in structure (e.g. actine or collagene), in mobility (e.g. myosine), in DNA conditioning (e.g. histones), in the catalysis of reactions (e.g. enzymes), etc. These molecules are needed in various locations in and outside the cell, and they don’t come into being where they are needed (Alberts et al., 2002).

In eukaryotes, after their biosynthesis, proteins have to follow a specific pathway that will allow them to be correctly folded and modified, before they can be delivered where they belong. This is called the secretory pathway. It’s a journey across the cell organelles that begins in the cytosol (Alberts et al., 2002).

1.1 - Targeting to the ER

In 1999 Blobel won the Nobel Prize in Physiology and Medicine for discovering that proteins have intrinsic signals that govern their transport and localization in the cell (Blobel and Dobberstein, 1975). This signal is a small peptide of fifteen to thirty amino-acids, with a hydrophobic core and a positive charge at its N-terminus and that is usually found at the N-terminus of secretory proteins (Cooper and Hausman, 2006). A signal anchor also exists for tail anchored proteins, whose charge distribution at the first transmembrane domain (TMD) determines the orientation of the protein insertion (Borgese and Fasana, 2011).

Once this sequence emerges from the ribosome, there is a pause in the translation. The signal recognition particle (SRP) binds to the signal peptide and forms a complex with the ribosome (Figure 1). The signal sequence is then recognized by the 54-kDa polypeptide of the SRP. This complex containing the nascent chain (NC), the ribosome and the SRP is then targeted to the endoplasmic reticulum (ER) membrane, where it binds to the SRP receptor, consisting of an α and a β subunit, next to a protein conducting channel, the Sec61 complex. The NC is inserted into this translocon, and the ribosome binds to the channel. Guanosine Triphosphate (GTP) binds to the GTP-binding G domain of the SRP and to both subunits of the SRP receptor, and its hydrolysis allows the release of both the SRP and its receptor, that then begin a new targeting cycle (Rapoport, 1992). It sometimes happens that the SRP and its receptor are not enough to allow the translocation of a protein. They can then use some help, such as the translocating chain-associating membrane protein (TRAMp) for membrane insertion (Gruss et al., 1999).

The signal peptide is then removed while the NC is emerging into the ER lumen by an enzyme, present on the lumenal side of the ER membrane, called signal peptidase. In the case of membrane proteins, it is possible that there is a stop transfer signal, that arrests the translocation, and the appropriate hydrophobic region migrates laterally into the membrane to become a TMD. Once in the ER lumen, proteins are taken over by chaperone molecules, that prevent inappropriate interactions between proteins, as well as protein aggregation, and promote protein folding (Vitale and Denecke, 1999).

The SRP targeting is a co-translational way to cross the ER membrane, but it may happen that proteins cannot be translocated co-translationally, because they are too small or their signal sequence is not located at their N-terminus (e.g. tail-anchored (TA) proteins). In the case of C-tail-

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anchored proteins, such as the yeast protein Sbh1p, the only targeting determinant is present at their C-terminus, and it is only available when the protein is released from the ribosome, thus these proteins have to be translocated post-translationally. Some of the post-translational targeting mechanisms have been discovered, and an example in yeast is the guided entry of tail-anchored proteins complex (GET complex; Borgese and Fasana, 2011). Get3 is an Adenosine Triphosphatase (ATPase) that binds specifically to the C-terminally localized hydrophobic domains on newly synthesized proteins. Get1 and Get2 are two membrane proteins localized on the ER membrane that serve as receptor, to which binds the Get3-TA complex. After the binding of this complex to the receptors, the TA protein is inserted into the membrane and can be further routed to another cell organelle when needed (Schuldiner et al., 2008).

Figure 1 - The SRP targeting cycle. This illustration shows the SRP targeting of nascent proteins to the ER

membrane. The signal sequence is first bound by the SRP (step 1), and the complex thereby formed binds to

the SRP receptor on the surface of the ER-membrane (step 2). The SRP is released by GTP hydrolysis and

starts a new cycle while the signal sequence is inserted into the translocon (step 3), where it is then cleaved

by SP (step 4), and the newly synthesized protein is finally released in the ER lumen (step 5) for further

modifications. Illustration from Cooper and Hausman, 2006.

1.2 - ER to Golgi trafficking

Once in the ER lumen, proteins can be subjected to several post-translational modifications, such as N-glycosylation by the Oligosaccharyl Transferase complex, disulfide bond formation by the protein disulfide isomerase (PDI) and addition of Glycosylphosphatidylinositol (GPI)-anchors. Those modifications are necessary for the correct folding of proteins by chaperones (Cooper and Hausman, 2006).

When proteins are fully folded and modified, they are incorporated into small transport vesicles (Figure 2). A coated protein complex (COPII) initiates the budding of this vesicle from the ER membrane, in association with other proteins. Those vesicles are released in the cytosol,

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proximal to the Golgi apparatus, and are transported to the ER-Golgi intermediate compartment (ERGIC) from where they are led to the cis-Golgi by microtubules and dynein, in COPI-coated vesicles. Some proteins need to be transported back to the ER, and this retrograde transport from Golgi to the ER is mediated by COPI coated-vesicles and require microtubules and kinesin. (Lord et al., 2013). Dynein and kinesin are motor proteins that hydrolyze ATP, allowing the anterograde movement on the microtubule (in the case of kinesin) or the retrograde movement (in the case of dynein) (Berg et al., 2002).

Once the COPII-coated vesicle is fused with the membrane of the cis-Golgi, a process named cisternal migration begins. Proteins move through several compartments (cis cisternae, medial cisternae and trans cisternae), where they may suffer additional post-translational modifications, such as N-linked glycosylation, O-linked glycosylation and phosphorylation. They finally arrive in the trans-Golgi network (TGN), in close proximity to the plasma membrane, where they are destined to be secreted (Lodish et al., 2000). Membrane proteins follow the same pathway to reach their final destination.

Figure 2 - ER to Golgi trafficking. This illustration represents the vesicle transport that occurs between the ER and the Golgi. Vesicles bud from the ER membrane, coated by COPII, and travel to the ERGIC (intermediate compartment), where they are then lead to the cis-Golgi by COPI coated vesicles (not shown). Proteins move to the trans-Golgi, and then to the TGN to be secreted. Retrograde transport to the ER is mediated by COPI-coated vesicles. Modified from Miller and Krijnse-Locker, 2008.

1.3 - Secretion and Exocyst

The proteins in the TGN are destined to be secreted. Those secretory proteins are sorted in

two types of vesicles, affecting the way they are secreted. In the case of proteins that have to be

continuously released from the cell, such as those that build the extracellular matrix, they are

transported into vesicles that directly move and fuse with the plasma membrane, allowing the

release of their content by exocytosis that is mediated by the exocyst complex. The other group

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contains proteins whose secretion has to be regulated, such as enzymes or hormones. In the TGN,

those proteins are sorted into secretory vesicles and stored inside the cell, waiting for a stimulus to

be secreted. This stimulus can be a neural or a hormonal signal, which leads to an increase of the

cytosolic Ca2+ concentration in the cytosol. This increase of Ca2+ triggers the fusion of the vesicle

membrane with the plasma membrane, and the release of the vesicle’s content (Lodish et al., 2000).

The exocyst is a 750-kDa complex, consisting in the following subunits: Sec3p, Sec5p, Sec6p,

Sec8p, Sec10p, Sec15p, Exo70p and Exo84p. The involvement of this complex in the exocytosis of

vesicles has first been identified in yeast (Lipschutz et al., 2003). Before the fusion with the plasma

membrane, vesicles containing the secretory proteins are recognized by the exocyst complex that

functions as a tethering factor for the vesicles at the plasma membrane (Toikkanen et al., 2003).

Sec4 is a small GTP-bound Rab protein found on the cytosolic side of the secretory vesicles’

membrane that interacts with the Sec15p subunit of the exocyst complex, presumably for specific

secretory vesicle recognition (Hutagalung and Novick, 2011; Lipschutz and Mostov, 2002). The

exocyst may form an initial connection between vesicle and plasma membrane, bringing the vesicle

close enough to promote SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein

receptor) complex formation and vesicle fusion and/or play a role in regulating SNARE assembly

(Heider and Munson, 2012).It has also been shown that the β subunit of the Sec61 translocon

interacts with the exocyst although they are located at opposite ends of the secretory pathway

(Toikkanen et al., 2003).

2. ACROSS THE MEMBRANE: PROTEIN TRANSLOCATION INTO THE ER

As described before, proteins can be translocated into the ER co- or post-translationally, according to their length and to the localization of their targeting sequence.

Several translocation complexes exist within the ER membrane, which are activated depending on which type of translocation will be used. The three main ones are the Sec61 complex, the Ssh1 complex and the Sec complex (Zimmermann et al., 2011).

2.1 - The Sec61 complex

The Sec61 complex is a protein translocation complex that is found in the ER membrane in eukaryotic cells. It has a homologue in bacteria and archea, named SecYEG, what shows that this complex is evolutionary conserved (Osbourne et al., 2005; Van den Berg et al., 2004; Wilkinson et al., 1996). It consists of three subunits. Sec61α, Sec61β and Sec61γ in mammals; Sec61p, Sbh1p and Sss1p in yeast (Figure 3); SecY, SecG and SecE in bacteria and archea. It is involved in co-translational protein translocation into the ER, and post-translational protein import when it is part of the heptameric Sec complex, forming the protein conducting channel (Finke et al., 1996; Harada et al., 2011; Kalies et al., 1994). This complex also plays a role in the ER-Associated Degradation (ERAD) pathway, as part of a large and dynamic complex consisting of an ER-resident ubiquitin ligase (Hrd1), the Sec61 channel, Sec63p and the proteasome 19S regulatory particle (RP) (Römisch, 2005).

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In addition to being able to translocate proteins through the ER membrane, the Sec61 complex also opens laterally, within the membrane, to allow the integration of hydrophobic transmembrane (TM) segments of a membrane protein (Osbourne et al., 2005).

Figure 3 - Sec61 complex. This figure represents the Sec61 complex in yeasts, consisting of three subunits: Sec61p, that forms the core of the channel, Sbh1p and Sss1p.

2.1.1 - The Sec61p Subunit

Sec61p is the main subunit of the Sec61 complex. Sec61p forms an aqueous channel whose opening and closing is tightly regulated (Mothes et al., 1994; Pilon et al., 1998). It is a protein that spans the membrane ten times, with its N- and C-termini in the cytosol. Its structure is closed by a lumenal plug domain and a hydrophobic constriction ring (Junne et al., 2010). These are proposed to prevent or reduce ion permeability during protein translocation (Gogala et al., 2014).

It is essential for the survival of the cell and with Sss1p, it forms the core of the translocation channel (Osbourne et al., 2005). It possesses two larges loops, L6 and L8, on the cytoplasmic side of the ER, and a large one, L7, on the lumenal side. L6 and L8 are important for ribosome binding during co-translational import and L7 is proposed to be interacting with lumenal chaperones and/or misfolded protein complexes in order to open the channel for the export of proteins for degradation in the cytosol (Tretter et al., 2013). The N-terminal region also seems to be particularly important to the function of this protein (Pilon et al., 1998).

2.1.2 - The Sbh1p Subunit

Sbh1p is a small 82 a.a. C-tail-anchored protein in the ER membrane with largely unstructured cytosolic domain and single α-helical TMD consisting of 25 a.a. and with 7 a.a. more presumably protruding into the lumen of the ER (Feng et al., 2007).

This protein is not essential to the survival of the cell. Nevertheless, the deletion of both SBH1 and SBH2 (its homologue that encodes a protein in the Ssh1 complex) leads to high temperature sensitivity (Soromani et al., 2012). It was also discovered that co-translational protein translocation is reduced in this mutant and that it manifests N-glycan trimming defects at permissive temperatures (Feng et al., 2007).

The cytosolic domain of Sbh1p can be phosphorylated, and Soromani et al. (2012) discovered that several phosphorylation sites might exist. Among the six phosphorylation sites suggested by bioinformatics tools, it turned out that their substitution with non-phosphorylable a.a., even in combination with the other, did not affect in any manner the function of Sbh1p. However, they discovered a new phosphor-acceptor site, a Thr at position 5, by mass spectrometry, and the further investigation on this site displayed interesting results. In fact, it seems that even when this site is mutated, the protein is not hypophosphorylated, suggesting that another substrate can be used by the same kinase, such as the Ser at position 3. This phosphorylation site at position T5, as well as

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the two Pro on its side evolved independently, in Saccharomyces cerevisiae (S. cerevisiae) and in mammals, but are not found in other yeasts, in other vertebrates, and in unicellular eukaryotes, suggesting that this site might be relevant for its function in the protein translocation, since this phosphorylation only takes place when the subunit is associated in the Sec61 complex.

The TMD of Sbh1p was also demonstrated as being an important domain of the protein. Feng et al. (2007) showed that the expression of the TMD alone in strains lacking both SBH1 and SBH2 was enough to rescue the temperature sensitivity. Moreover, it was proven to be sufficient to ensure Sbh1p functions, and its interaction with other partners, such as the SP, the ribosome (Feng et al., 2007) and the exocyst (Toikkanen et al., 2003).

These data indicate that Sbh1p acquired function independent of protein translocation into the ER or regulatory function that coordinate translocation with other events (Feng et al., 2007).

Since this subunit is not essential, it was suggested that its function may be to enhance the speed of the translocation or the efficiency of the targeting (Soromani et al., 2012), but this process has been demonstrated so far.

2.1.3 - The Sss1p Subunit

Sss1p is a 9-kDa TA protein that together with the Sec61p subunit, forms the core of the Sec61 complex, and thus is essential for cell viability (Falcone et al., 2011).

The C-terminal end of the hydrophobic sequence of this protein plays a role in the proper assembly of the Sec61 and Sec complexes, as it was shown that mutations in it compromises the stability of these complexes, resulting in a severe defect in the function of the Sec61 complex and leading to a complete reorganization of the ER structure (Falcone et al., 2011). Moreover, its cytoplasmic region has been shown to interact with the OST, like the TMD of Sbh1p (Chavan et al., 2005).

The precise mechanism of action of this protein is largely unknown, but several hypotheses have been proposed. It may help to maintain the membrane permeability barrier, facilitate the oligomerization of the Sec61 complex and it may clamp together the two halves of the Sec61p subunit (Falcone et al., 2011).

2.2 - The Ssh1 Complex

The Ssh1 complex is a trimeric complex that is structurally related to the Sec61 complex. It consists of three subunits: Ssh1p, Sss1p and Sbh2p (a homologue of Sbh1p) (Finke et al., 1996). It is involved exclusively in co-translational translocation in the ER (Soromani et al., 2012).

It has also been proposed that it is involved in the ERAD pathway (Feng et al., 2007). As the Sec61 complex, the Ssh1 complex also interacts with membrane bounds ribosomes, but does not associate with the Sec62/63p complex to form a heptameric complex (Finke et al., 1996).

This complex is not essential for cell viability, but it is required for normal growth rates. It is found in equal abundance as the Sec61 complex in yeast microsomes, which is the reason why it is thought that both can transport distinct proteins, or that two different targeting pathways are used. An alternative hypothesis is that having two co-translational translocation systems could allow the cell to regulate co- and post-translational pathways independently from each other (Finke et al., 1996).

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2.3 - The Sec Complex

The Sec complex is a heptameric complex that mediates post-translational import into the ER of proteins with moderately hydrophobic signal sequences in yeast. It consists of the association between the tetrameric Sec62/Sec63 complex (containing Sec63p, Sec62p, Sec71p and Sec72p) and the Sec61 complex (Deshaies et al., 1991; Osbourne et al., 2005). The energy required for post-translational translocation comes from the hydrolysis of ATP by the Kar2p chaperone molecule in the ER lumen, a homologue of the mammalian BiP (Pilon et al., 1998).

Proteins using the post-translational pathway are kept in a translocation competent conformation by molecular chaperones, which also play a role in the targeting to the Sec complex (Falcone et al., 2011). The Sec62/63 complex plays a crucial role in targeting of SRP-independent protein substrates to the protein conducting channel. It is also proposed to play an important role in the assembly of the post-translocon (Harada et al., 2011).

3. POST-TRANSLATIONAL MODIFICATIONS: PROCESSING IN THE ER

Since their first a.a. emerge in the ER lumen, proteins are subject to several events, that will prepare them for their journey through the secretory pathway.

The proteins first undergo some modifications (N-linked glycosylation, disulfide bond formation, addition of GPI-anchors, etc). Their folding is then controlled by a process called ER Quality Control (ERQC) and they are sent to further organelles or retained in the ER to be led to the cytosol for degradation. The ER-Associated Degradation pathway sends the misfolded or unfolded proteins into the cytosol for degradation by the 26S proteasome, but when it is not efficient, it leads to stress in the ER, and to a specific response called Unfolded Protein Response (UPR; Lodish et al., 2000).

3.1 - Evolving into glycoprotein

3.1.1 - N-linked glycosylation

As soon as a glycosylatable site in the NC emerges in the ER lumen, an oligosaccharide is transferred to the NH2 group of an asparagine in a selected Asparagine-X-Serine/Threonine (Asn-X-Ser/Thr) sequence, with X being any a.a. except Proline (Pro; Aebi, 2013).

The precursor oligosaccharide is formed by a fourteen sugar long chain constisting of two residues of N-acetylglucosamine (GlcNAc), nine residues of mannose (Man) and three residues of glucose (Glc) and is anchored in the ER membrane by a lipid molecule called dolichol diphosphate (Dol-P-P; Herscovics and Orlean, 1993). This oligosaccharide is transferred to the target Asn in a single enzymatic step catalyzed by membrane bound enzyme called Oligosaccharyl Transferase, which has its active site exposed to the lumenal side of the ER membrane (Silberstein and Gilmore, 1996).

Since most of the proteins are imported in a co-translational way into the ER, N-linked oligosaccharide are almost always added during protein biosynthesis (Alberts et al., 2002).

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Although N-linked glycosylation is the most common type of glycosylation, oligosaccharides can be linked to the hydroxyl group on the side chain of a serine, threonine or hydroxylysine (Hyl) a.a. This process is called O-linked glycosylation and happens in the Golgi (Alberts et al., 2002).

3.1.2 - Oligosaccharyl Transferase Complex

The Oligosaccharyl Transferase Complex (OST) consists of the following subunits: Ost1p, Ost2p, Stt3p, Wbp1p and Swp1p, which are encoded by essential genes, and Ost3p, Ost4p, Ost5p and Ost6p, which are non-essential, but required for optimal activity of the enzyme (Chavan and Lennarz, 2006; Silberstein and Gilmore, 1996).

In yeast, two isoforms of this complex have been found. One is made of eight subunits, including Ost3p, but not Ost6p, and the other contains Ost6p, but not Ost3p. It has been shown that those two complexes interact with two different translocation complexes: Ost3p interacts specifically with the Sbh1p subunit of the Sec61 complex, and Ost6p interacts specifically with the Sbh2p subunit of the Ssh1 complex (Yan and Lennarz, 2005). A severe underglycosylation phenotype in soluble and membrane proteins has also been identified for mutants lacking both OST3 and OST6 (Chavan and Lennarz, 2006).

In addition to its interaction with Sbh1p or Sbh2p, some of the subunits of the OST also interact with other components of the translocon. Stt3p, for example, was found to interact only with Sec61p, whereas Ost4p interacts with all if the subunits of the translocon. Wbp1p interacts strongly with Sec61p and Sbh1p, and weakly with Sss1p. An interaction with Sec61p and either Sbh1p or Sss1p was also found for the Ost1p, Ost2p and Swp1p subunits, whereas Ost5p and Ost6p failed to interact with Sbh1p or Sss1p (Chavan et al., 2005). Besides those multiple interactions between the two complexes, it has also been demonstrated that the OST physically binds to the 60S ribosomial subunit (Harada et al., 2009).

Each subunit of this complex has a specific function, and a model for the sequential steps of the glycosylation has been proposed. Ost3p and Ost6p determine the association of a specific isoform to a specific translocon, and Ost4p helps in the association of both complexes. Ost1p guides the unfolded protein to the active site of the complex while Wbp1p presents the Dol-P-P-oligosaccharide to the catalytic subunit of the OST. Stt3p scans the NC for glycosylatable sites and catalyses the N-linked glycosylation of polypeptides, which reduces the affinity to the complex, allowing the dissociation from this protein (Chavan and Lennarz, 2006).

3.2 - More post-translational events

Besides the N-Glycosylation of the nascent polypeptide chain, several modification and events happen in the ER lumen, starting from the cleavage of the signal peptide that allows the targeting to the ER by the SP on the lumenal side of the ER membrane (Alberts et al., 2002).

Among the modifications undergone by the protein after its translocation, there is the formation of disulfide bonds. These bonds are essential for the stability of the tertiary and quaternary structure of many proteins. The oxidation of the free sulfhydryl (SH-) groups of cysteines (Cys) is catalyzed by PDI (Alberts et al., 2002; Lodish et al., 2000).

The addition of GPI-anchors to membrane proteins is another modification that the protein has to go through. GPI-anchors are essential for the survival of S. cerevisiae for example, because they are used to target certain mannoproteins for covalent incorporation into the β-glucan cell wall. A precursor is pre-assembled in the ER membrane, before being attached to the newly synthesized

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protein in the ER lumen and undergo lipid remodeling and/or carbohydrate side-chain modification in the ER and the Golgi (Varki et al., 2009).

All those modifications, together with folding enzymes and different types of chaperone molecules, help the folding of newly synthesized proteins, the prevention of inappropriate interactions with other proteins (Braakman and Hebert, 2013).

3.3 - Protein inspection - ER Quality Control

Despite the process describe above, it can happen, that proteins are not able to reach their native conformation (corresponding to the most energetically favorable state). These kind of proteins, which are immature, misfolded or unfolded, are problematic because they are toxic for the cell, as they cannot ensure their function correctly and can also aggregate (Ellgaard and Helenius, 2003). This misfolding can be caused by mutations, substoichiometric amounts of a binding partner or a shortage of chaperone availability (Ruggiano et al., 2014). The role of the ERQC is to recognize the misfolded proteins, and send them for further folding or degradation (Buchberger, 2014).

In order to recognize those proteins, the cell uses sensor molecules, such as the molecular chaperone Kar2p, calnexin, calreticulin, glucose-regulated protein 94 (GRP94) and the thiol-disulfide oxidoreductases PDI and ERp57 (Ellgaard and Helenius, 2003). BiP (Kar2p in yeast) is an ER-resident chaperone molecule, which is a members of the Hsp70 family. It recognizes incorrectly folded proteins by binding to exposed a.a. sequences that should be buried in the interior of correctly folded polypeptide chains. In addition to its role in protein folding, it also plays a role in the ERQC by preventing proteins from aggregating and retaining misfolded or unfolded proteins in the ER (Alberts et al., 2002; Lodish et al., 2000)

The quality control (QC) is divided in a primary and a secondary QC. The primary QC works at a general level, on all proteins, whereas the secondary QC is reserved for selected categories of proteins, and is more specific. The general QC distinguishes between native and non-native proteins basing on the exposure of hydrophobic regions, unpaired Cys residues and the tendency to aggregate. It frequently happens that one or more of the sensor molecules bind to a protein with even minor deviation from the native conformation (Ellgaard and Helenius, 2003).

A well-characterized primary QC is the calnexin/calreticulin cycle (Figure 4). This cycle controls the quality of glycoproteins, by keeping uncorrectly folded ones in the ER until they reach their native conformation, or targeting them to for degradation. Glucosidase I and II (Gls1/2p) remove two glucose residues from the oligosaccharide, which leaves a monoglucosylated glycoprotein. This protein binds to the globular lectin domain of celnexin or calreticulin. Both form complexes with ERp57 and calreticulin-bound glycoproteins. Gls2p hydrolyses the glucose residue from the monoglucosylated glycan, thus dissociating the latter from calnexin or clareticulin. The correctly folded protein is then released from the ER to the Golgi in a transport vesicle, whereasl misfolded proteins are reglucosylated by an UDP-glucose:glycoprotein glucosyltransferase (GT) to enter the cycle again (Ellgaard and Helenius, 2003; Hammond and Helenius, 1995; Ware et al., 1995).

3.4 - The good, the bad and the misfolded - ER-Associated Degradation

It is estimated that 30% of the proteins in eukaryotic cells are misfolded during their biosynthesis and degraded (Schubert et al., 2000). These misfolded proteins can form toxic aggregates in the cell, and this is why cell have developed a way to eliminate them: the ERAD (Figure 5; Römisch, 2004).

18

Once they are recognized as un- or misfolded, these proteins are targeted for degradation in the cytosol. In order to be retrotranslocated in the cytosol, proteins have to remain in a soluble form, helped by Bip/Kar2p and other proteins (Römisch, 2005). This retrotranslocation is mediated by Sec61p in association with other proteins and complexes (Wiertz et al., 1996).

Figure 4 - Calnexin/Calreticulin cycle. This is the specific cycle for folding and degradation of glycoproteins. After the addition of an N-glycan to the polypeptide (1), two glucosidases, Gls1 and 2, remove two glucose residues from the oligosaccharide (2). This monoglucosylated oligosaccharide then binds to the globular lectin domain of calreticulin or calnexin (not shown), itself bound to ERp57 (3). The hydrolysis of the remaining glucose residue by Gls2 allows the dissociation of the protein from the calreticulin/calnexin complex (4), and if the protein is correctly folded, it is led to an ER exit site, for further processing in the secretory pathway (5). In case the protein is still not correctly folded, an UDP glucose:glycoprotein GT adds a new glucose residue to the glycan, so that it can enter the cycle again, until proper folding (6). Sometimes, the protein fails to be correctly folded. In this case, an α1,2-mannosidase remove a mannose from the oligossacharide chain (7), so that it can be targeted by the EDEM and retrotranslocated into the cytosol for degradation (8). Adapted from Ellgaard and Helenius, 2003.

19

Proteins that have to be degraded need to be ubiquitinated. Ubiquitin is a 76 a.a. long protein which is covalently attached to the target protein through the sequential action of activating (E1), conjugating (E2) and ligase (E3) enzymes (Pickart, 2001; Vembar and Brodsky, 2008). This event takes place in the cytosol, after the misfolded protein has been retrotranslocated (Römisch, 2005). The best characterized ubiquitylation system is mediated by the E3 ligase complexes Hrd1p and Doa10p (Hampton, 2002). Which complex is used for which protein is determined by the localization of the misfolded protein, and in case of transmembrane proteins, the localization of their misfolded domain: when it is localized in the cytoplasmic domain (ERAD-C substrates), the degradation occurs via the Doa10 complex, whereas in the case of lumenal (ERAD-L substrates) or intramembrane (ERAD-M substrates) misfolded domains, proteins are degradated via the Hrd1 complex (Ruggiano et al., 2014; Vembar and Brodsky, 2008). Ufd2p, Cdc48p, Rpn10p and Rpt5p (subunits of the 19S RP of the proteasome), as well as Rad23p and Dsk2p mediate the polyubiquitylation of the protein, and possibly mediate the transport to the proteasome proteolytic core (Römisch, 2005).

The degradation of glycoproteins happens in a different way (Figure 4). A mannose in the middle branch of the N-linked oligosaccharide is removed by the α1,2-mannosidase I, leading to an association with ER degradation-enhancing 1,2-mannosidase-like protein (EDEM), localized on the ER membrane (Ellgaard and Helenius, 2003; Vembar and Brodsky, 2008).

Proteins that are correctly folded can also be targeted by the ERAD. Their degradation is highly regulated and can only happen in the presence of a specific signal. 3-hydroxy-3-methylglutaryl acetyl-coenzyme-A reductase (HMGR) and squalene monooxygenase (SQLE or Erg1 in yeasts) are two enzymes of the sterol biosynthetic pathway, which suffer this type of degradation. Indeed, this is part of a feedback inhibition system in order to prevent the accumulation of sterol metabolites, which are toxic for the cell (Foresti et al., 2013).

3.5 - Stress in the ER and Unfolded Protein Response

The inactivation of ERAD results in an accumulation of misfolded proteins in the lumen or membrane of the ER, inducing ER-stress. This ER-stress activates an intracellular signal transduction pathway, called UPR (Walter and Ron, 2011), whose role is to maintain protein-folding capacity in the ER (Korennykh and Walter, 2012).

The UPR involves one sensor in yeast, called Ire1, and three in higher eukaryotes, named Ire1, PERK and ATF6 (Korennykh and Walter, 2012).

Ire1 is a kinase and consists of a N-terminal sensor domain in the ER lumen and a CDK2-like Ser/Thr kinase domain fused to an unique C-terminal ribonuclease (RNase) domain in the cytosol. When an unfolded/misfolded protein binds to its sensor domain, it activates the RNase domain, that further cleaves HAC1 mRNA at two unconventional sites (Cox and Walter, 1996). The two resulting exons are associated by tRNA ligase, and this induces the translation of functional Hac1 transcription factors. After entering the nucleus, the transcription factors activate the transcription of UPR target genes, thus increasing the ER protein-folding capacity to the cell (Korennykh and Walter, 2012).

Prolonged activity of the UPR indicates that ER-stress cannot be moderated, and induces apoptosis (Tabas and Ron, 2011).

20

Figure 5 - ERAD Pathway. The first step in the ERAD is the recognition by sensor molecules of misfolded proteins (1). Those misfolded proteins are then targeted to ligase enzymes (2), depending on the location of the misfolded domain (Doa10 complex for cytosolic, Hrd1 for lumenal and intramembrane misfolded proteins), in order to be retrotranslocated in the cytosol (3). Once in the cytosol, these proteins are oligo-ubiquitylated by those ubiquitin ligase complexes (4), polyubiquitylated by a protein complex (5), and may then be further led by Rad23p and Dsk2p to the 26S proteasome (6) for degradation (7). Modified from Meusser et al., 2005.

4. AIM OF THIS STUDY

As it was described in the work of Soromani et al. (2012), T5 and S3 are two important phosphorylation sites discovered on the cytosolic domain of Sbh1p. The mutation of Thr to Ala at position 5 results in the destabilization of Sbh1p, but not in a hypophosphorylated protein. This result suggests that the kinase responsible for the phosphorylation of the T5 was able to use other residues as substrate, and the Ser at position 3 was proposed as a candidate for phosphorylation by this kinase. The T5 phosphorylation site on Sbh1p evolved twice independently (in S. cerevisiae and mammals) and is thus suggested to play a particularly important role, considering also that it is only phosphorylated when part of the Sec61 complex.

21

Another domain of Sbh1p has been shown to have an important role: the TMD (Feng et al., 2007). In fact, the expression of the TMD alone affects neither the functionality of the protein nor its interaction with the Sec61 complex. The import of N-glycosylated α-factor precursor (3gpαf) by the Sec61 complex was investigated in order to detect the defects in the functionality of Shb1p due to the lack of its cytosolic or transmembrane domains. This led to the discovery that Δsbh1Δsbh2 cells show a reduced level of Msn1p due to constitutive translocation defects, further leading to a reduced efficiency in the N-glycan trimming by glucosidase and mannosidase.

Following this finding that the TMD has an essential role, Zhao and Jäntti (2009) decided to generate random mutations in the latter, in order to characterize the function of this domain. This random mutagenesis led to the discovery that the TMD can be inactivated by the combination of two point mutations: P54S and V57G.

This study is based on these findings, and it is aimed at the investigation of the influence of these mutations in the phosphorylation sites and mutations in the TMD on the import of Gls1p, one of the two other proteins that trims N-glycan for the ERQC.

As Sec61 is part of a bigger complex that allows the retrotranslocation of proteins into the cytosol, these mutations might have affected the proper function of the ERAD, thus a part of this work was also aimed at the study of possible defects caused by these mutations in the very important process that is the ERAD.

22

II. Materials and Methods

1. MATERIALS

1.1 - Laboratory equipment, chemicals, reagents and consumables

Table 1 - Chemicals and reagents used in this study

Supplier Products

AGFA Healthcare GmbH G153 Developer A/B

G354 Rapid Fixer

AnalaR® NORMAPUR® (part of VWR® International GmbH)

Sodium Chloride

Glycine

Acetic Acid 100%

Hydrochloridric Acid

AppliChem GmbH

D(+) Glucose anhydrous

Sodium Dodecyl Sulfate

Cycloheximide

Tween® 20

Ampicillin Sodium Salt

Sodium Dihydrogen Phosphate

Becton, Dickinson and Company

Yeast Nitrogen Base without amino-acids

Yeast Nitrogen Base without amino-acids and Ammonium Sulfate

Bacto™ Casamino-acids

Bio-Rad Laboratories, Inc. 30% Acrylamide/Bis solution, 37.5:1

CalbioChem® (part of Merck Millipore) Digitonin

Carl Roth® GmbH and Co. KG Sodium Azide

Ammonium Persulfate

23

Carl Roth® GmbH and Co. KG

Sorbitol

Phenylmethylsulfonyl Fluoride

Yeast Extract

Peptone

Agar, Agar Kobe I

Triton X-100

Polyethyleneglycol 4000

Fisher Scientific (part of Thermo Fisher Scientific)

Methanol

Formedium™

Complete Amino-acid Drop-out : -Leu (Kaiser Mixture)

Complete Amino-acid Drop-out : -Leu/-Ura (Kaiser Mixture)

Complete Amino-acid Drop-out : -Ura (Kaiser Mixture)

GE Healthcare Protein-A Sepharose™ CL-4B

Grüssing GmbH Glycerol

Life Technologies (Part of Thermo Scientific)

Novex® MES Buffer NuPage

NuPage® 4-12 % Bis-Tris Gel 1.00 mm x 10 wells (Novex®)

Novex® MOPS Buffer NuPage

Merck Millipore Magnesium Chloride

Perkin Elmer® Inc.

EXPRE35S35S Protein Labeling Mix, [35S]-, 50 mM Tricine (pH 7,4), 10 mM 2-

Mercaptoethanol

[Methyl-14C] Methylated Protein Molecular Weight Marker

Roche GmbH Complete ULTRA Tablets, Mini, EDTA-free,

EASYpack

24

Sigma-Aldrich®

Trizma Base®

Dimethylsulfoxyde

Lithium Acetate

Deoxyribonucleic Acid Sodium Salt from Salmon Testes

Trypton

Ethanol

Tetramethylethylenediamine

Bromophenol Blue sodium salt for molecular biology, for electrophoresis

Ethylenediaminetetraacetic Acid

Urea

Dithiothreitol

Sucofin® Skimmed Milk Powder

Thermo Scientific

Detection Reagent 1 Peroxide Solution

Detection Reagent 2 Luminol Enhancer Solution

Restore™ Western Blot Stripping Buffer

Super Signal® West Dura Stable Peroxide Buffer

Super Signal® West Dura Luminol/Enhancer Solution

PageRuler Prestained Protein Ladder

Zentrales Chemikalien Lager Disodium Hydrogen Phosphate

25

Table 2 - Laboratory equipment used in this study

Company Product

Amersham Pharmacia Biotech Hypercassette™

Berthold technologies GmbH & Co. KG LB124 Contamination Monitor

Bio-Rad Laboratories, Inc.

PowerPac™ HC Power Supply

PowerPac™ 1000 Power Supply

PowerPac™ 3000 Power Supply

ChemiDoc™ XRS

Trans-Blot® Electrophoretic Transfer Cell

Model 583 Gel Dryer

Bio Spec Products Inc. Mini-Beadbeater-24

CAWO Solutions CAWOmat 2000 IR

Denver Instruments GmbH Ultra-basic 10 pH/mV Meter

Eppendorf AG

Minispin® Centrifuge

Thermomixer® Compact

Thermomixer® 5436

Microcentrifuge 5415R

Multipette® Plus

GE Healthcare

Image Eraser

Amersham Imager 600 RGB

Typhoon™ Trio Variable Mode Imager

Storage Phosphor Screens

Exposure Cassettes

Ultrospec 2100 pro UV/Visible Spectrophotometer

Gilson N10478B, N23112C, EA56493, GC54948

26

Heraeus Instruments GmbH Hot Chamber

Hirschmann® Laborgeräte Pipetus® A41585G

IKA®-Werke GmbH & Co. KG RTC Basic (Laboratory overhead stirrer)

Infors HT Multitron Standard Incubation Shaker

Invitrogen™ (Life Technologies part of ThermoFisher Scientific Inc.)

X-Cell Surelock™ Mini-Cell (Novex®)

Life Technologies Mini Gel Tank

LTF Labortechnik GmbH and Co. KG Unigeldryer 3545D

Merck Millipore Milli-Q® Integral Water Purification System

neoLab Migge Laborbedarf Rotary wheel

ST5 (shaker)

New Brunswick Scientific (part of Eppendorf AG)

Innova 4230 refrigerated incubator shaker

Peqlab NanoDrop 2000c Spectrophotometer

Rigal Bennett Luckham R100 Rotatest shaker

Sartorius AG Balance

Precision Balance

Scientific Industries Inc. Vortex-Genie® 2

Scotsman® Ice Systems AF80 Flake Ice Machine

Sigma-Aldrich® SIGMA 2-16P Refrigerated Centrifuge

Stuart® (part of Bibby Scientific Limited) Mini orbital shaker SSM1

Systec GmbH DX-150 Autoclave

VB-55 Autoclave

Vacuubrand GmbH+Co Chemistry Vacuum System MZ 2C NT +2AK

VARIO®-SP Diaphragm Pump MZ 2 VARIO-SP

Zeiss West Germany Binocular Light Microscope

27

Table 3 - Consumables used in this study

Company Product

B. Braun Melsungen AG Surgical Disposable Scalpel

Sterican® hypodermic needle Gr1/Gr16

Becton, Dickinson and Company Plastipak™ Sterile Syringe Luer-Lock™

20 mL

Bio-Rad Laboratories, Inc. Nitrocellulose Membrane (0,2 µM, 0,45 µM

pore size)

Biozym Scientific GmbH Surphob® Tips 1250 µL and 200 µL

Carl Roth® GmbH and Co. KG

Aluminium foil

Cover Glass 22 mm x 22 mm

Microscope Slides

Disposal bags

Costar® Stripette® 25 mL Serological Pipettes

Duran® Group

Graduated Cylinders

Erlenmeyer flask

Beaker

Solution Bottles

Eppendorf AG 2,5 mL, 5 mL, 12,5 mL Combitips

Fujifilm Fuji Medical X-Ray Film (Super RX)

GE Healthcare Whatman™ Paper

Greiner Bio-one International GmbH

Cellstar ® 10 mL serological pipettes

Cellstar® 14 mL and 50 mL falcons

Ultratips 1250 µL and 200 µL

Henke Sass Wolf 60 mL Norm-Ject® Luer-Lock™ Sterile

5 mL Soft-Ject® Luer Sterile

28

Life Technologies 1.0 mm/1.5 mm Gel Cassettes (Novex®)

Nalgene™ Labware (part of Thermo Scientific) 250 mL and 500 mL Rapid-Flow™ Filters

0,2 µm aPES membrane

neoLab Migge Laborbedarf Blotting Paper Sheets 200 x 200 mm

330 g/m2

Pechiney Plastic Packaging Parafilm “M”®

Sarstedt

10 µL Inoculation Loops

5 mL Serological Pipettes

Petri dishes

Micro-tubes 1,5 mL, 2,0 mL

Plastic Cuvettes

Sartorius AG Minisart® Sterile Syringe Filters (0.2µm pore

size)

Sigma-Aldrich

5 ml, 10 mL, 25 mL Volac® Graduated Glass Pipettes

Glass beads, acid washed

VWR®

Latex gloves

500 mL Bottles and Filter Upper Cup

0,2 µm PES membrane sterile

Sterile syringe Filter 0,2 µm Cellulose Acetate

1.2 - Media and Buffers

Table 4 - Composition of the media used in this study

Name Composition

LB Medium (+Agar)

1% Trypton from Casein

0,5% Yeast Extract

0,5% Sodium Chloride (NaCl)

29

(1,5% Agar)

100 mg/L Ampicillin

YPD Medium (+Agar)

2 % Peptone from Casein

1 % Yeast Extract

(2 % Agar)

2% Glucose

Drop-Out Medium:

-LEU (+Agar)

(2 % Agar)

1,622 g/L Complete Amino-acid Drop-out: -Leu

6,7 g/L Yeast Nitrogen Base (YNB) without amino-acids (a.a)

2 % Glucose

Drop-Out Medium:

-LEU/-URA (+Agar)

(2 % Agar)

1,546 g/L Complete Amino-acid Drop-out: -Leu/-Ura

6,7 g/L YNB without (w/o) a.a

2 % Glucose

Drop-Out Medium:

-URA (+Agar)

(2 % Agar)

1,926 g/L Complete Amino-acid Drop-out: -Ura

6,7 g/L YNB w/o a.a.

2 % Glucose

Minimal Medium

0,2% Casamino-Acids (CAA)

5% Glucose

Auxotrophic Amino-acids (ref. Annex 1)

6.7 g/L YNB without a.a

Table 5 - Composition of the solutions for yeast transformation

Name Composition

10x LiAc pH 7,5 1 M Lithium Acetate (LiAc)

Acetic Acid to optimize pH to 7,5

30

Table 6 - Composition of the solutions for cell extract preparation

Name Composition

DTT 1 M Dithiothreitol (DTT)

2x Sample Buffer

50 mM Tris-HCl pH 7,5

5 % Sodium Dodecyl Sulfate (SDS)

20 % Glycerol

2 % Bromophenol Blue

Table 7 - Composition of the solutions for SDS-PAGE and Western Blot

Name Composition

10x Tris-Glycine 25 mM Trizma Base

2 M Glycine

Transfer Buffer 1x Tris-Glycine

2 % Methanol (MetOH)

10x TE pH 7,5


10 mM Ethylenediaminetetraacetic Acid (EDTA)

Hydrochloridric Acid (HCl) to optimize pH to 7,5

1M Tris-HCl pH 7,5 121,14 g/L Trizma Base

HCl to optimize pH to 7,5

50% PEG4000-solution 500 g/L Polyethyleneglycol (PEG) 4000

LiAc/TE-solution 1x LiAc pH 7,5

1x TE pH 7,5

PEG solution

1x LiAc pH 7,5

1x TE pH 7,5

40% PEG4000-solution

31

1 % SDS

10x Tris-Buffered Saline (TBS) pH 7,4

0,5 M Tris HCl

1,5 M NaCl

HCl to optimize pH to 7,4

TBS-T 1x TBS pH7,4

1 % Tween-20

Milk Buffer

1x TBS pH 7,4

5 % non-fat dry milk

1 % Tween 20

5 mM Sodium Azide (NaN3)

10x SDS-Running Buffer

250 mM Tris-HCl

2 M Glycine

1 % SDS

SDS-PAGE Gel 7,5%

7.5% Resolving Gel

5% Stacking gel

4,7925 mL Water (H2O)

2,5 mL 30% Acrylamide

2,5 mL 1,5 M Tris-HCl pH 8,8

0,1 mL 10% SDS

0,1 mL 10% Ammonium Persulfate (APS)

7,5 µL Tetramethylethylenediamine (TEMED

2,1 mL H2O

0,5 mL 30% Acrylamide

0,38 mL 1 M Tris-HCl pH 6,8

30 μL 10% SDS

30 μL 10% APS

3 μL TEMED

Table 8 - Composition of the solutions for pulse experiments

Name Composition

Labelling Medium 5 % Glucose

Autotrophic a.a

32

6,7 g/L YNB without Ammonium Sulfate and a.a

Tris-Azide 20 mM Tris-HCl pH 7,5

20 mm NaN3

Resuspension Buffer


10 mM DTT

20 mM NaN3

Lysis Buffer


2 % SDS

1 mM DTT

1 mM Phenylmethanesulfonyl Fluoride (PMSF)

IP Buffer

150 mM NaCl

1 % Triton X-100 (TX-100)

0,1 % SDS

1mM PMSF


2 mM NaN3

Urea Wash

2 M Urea

200 mM NaCl

1 % TX-100


2 mM NaN3

ConA Wash

500 mM NaCl

1 % TX-100


2 mM NaN3

Tris-NaCl Wash

50 mM NaCl


2 mM NaN3

Fixing Solution 1 10 % Acetic Acid

33

40 % MetOH

2 % Glycerol

Fixing Solution 2 50 % MetOH

1 % Glycerol

Table 9 - Composition of the solutions for native immunoprecipitation

Name Composition

Lysis Buffer

200 mM Sorbitol

100 mM NaCl

25 mM Sodium Phosphate Buffer pH 7,4

1 mM Magnesium Chloride (MgCl2)

1 mM PMSF

1x Protease Inhibitor Cocktail

10% Glycerol

Sodium Phosphate Buffer pH 7,4

80% 1 M Disodium Hydrogen Phosphate (Na2HPO4)

20% 1 M Sodium Dihydrogen Phosphate (NaH2PO4)

1.3 - Bacterial and yeast strains

Table 10 - Escherichia coli (E.coli) strains used in this study

Strain Genotype Source

DH5α

F endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15

Δ(lacZYA-argF)U169, hsdR17(rK- mK

+), λ–

Hanahan, 1983

XL-1 Blue recA1 endA1 gyrA96 thi-1 hsdR17

supE44 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)]

Stratagene: cat.#200249

KRB319 pDN431 (CPY*HA; URA3) Ng et al., 2000

34

Table 11 - S. cerevisiae strains used in this study

Strain Genotype Source

KRY293 leu2-3,112 ura3-52 seb::URA3,

MATα J.H. Toikkanen

KRY294 leu2-3,112 ura3-52 seb2::G418R,

MATa J.H. Toikkanen

KRY585 leu2-3, 113 ura3-52, MATa P. Novick, J. Jäntti

KRY588 seb1::KanMx seb2::hphMx leu2-

3,112 ura3-52 GAL+, MATa D. Feng, J. Jäntti

KRY879 ade2-1 ura3-1 his3-11,15 leu2-

3,112 trp1-1 can1-100 der1::natNT2

C. Servas (Römisch Lab)

KRB320 pDN436 (CPY*HA; LEU2) Ng et al., 2000

KRB536 pRS415 (in XL-1 Blue) Sikorski et al., 1987

KRB689 SEB1pRS415 (in DH5α) C. Soromani

KRB746 S3ApRS415 (in DH5α) C. Soromani

KRB747 T5ApRS415 (in DH5α) C. Soromani

KRB987 YEpSBH1TM(P54S,V57G) (in DH5α) Zhao et al., 2007

KRB1032 pRS415 SBH1 S3A/T5A (in XL-1 Blue) J. Simon

KRB1046 pRS415 SBH1 S3A TMDM (in XL-1

Blue) J. Simon

KRB1047 pRS415 SBH1 T5A TMDM (in XL-1

Blue) J. Simon

KRB1048 pRS415 SBH1 S3A/T5A TMDM (in

XL-1 Blue) J. Simon

KRB1050 pRS415 SBH1 TMDM (in XL-1 Blue) J. Simon

35


3,112 ura3-52 GAL+, MATa [pRS415 SBH1 S3A/T5A]

J. Simon


3,112 ura3-52 GAL+, MATa [pRS415 SBH1 S3A TMDM]

J. Simon


3,112 ura3-52 GAL+, MATa [pRS415 SBH1 T5A TMDM]

J. Simon

KRY1021

seb1::KanMx seb2::hphMx leu2-3,112 ura3-52 GAL+, MATa

[pRS415 SBH1 S3A/T5A TMDM]

J. Simon


3,112 ura3-52 GAL+, MATa [pRS415 SBH1 TMDM]

J. Simon

1.4 - Plasmids

Table 12 - Plasmids used in this study

Plasmid Marker / Selection Carrier Strain Reference/Source

pRS415 LEU2 / Amp KRB536 Sikorski et al. (1987)

pDN431-CPY*HA URA3 / Amp KRB319 Ng, et al. (2000)

pDN436-CPY*HA LEU2 / Amp KRB320 Ng, et al. (2000)

S3ApRS415 LEU2 / Amp KRB746 C. Soromani

SEB1pRS415 LEU2 / Amp KRB689 C. Soromani

T5ApRS415 LEU2 / Amp KRB747 C. Soromani

YEpSBH1TM (P54S,V57G) LEU2 / Amp KRB987 Zhao et al. (2007)

pRS415 SBH1 S3A/T5A LEU2 / Amp KRB1032 J. Simon

pRS415 SBH1 S3A TMDM LEU2 / Amp KRB1046 J. Simon

pRS415 SBH1 T5A TMDM LEU2 / Amp KRB1047 J. Simon

36

1.5 - Antibodies

Table 13 - Antibodies used in this study

Antibody Protein detected Molecular

Weight of the Protein

Use and Working Dilution

Source

anti-Cdc48p (rabbit)

Cell Division Cycle

92 kDa WB – 1:1250 N. Zheng

anti-CPYp (rabbit)

Carboxypeptidase Y

pCPY = 59 kDa; p1CPY = 67 kDa; p2CPY = 69 kDa; mCPY = 61 kDa

WB - 1:3000 KB Römisch

anti-Gls1p (rabbit)

Glucosidase 1 96 kDa WB – 1:2000

IP – 1:100 A. Shibuya

anti-Rpn12p (rabbit)

Proteasome Regulatory

Particle Non-ATPase

35 kDa WB – 1:2000 K. Kalies

anti-Sbh1p (rabbit)

Sec61 beta homolog 1 (N-

terminus)

9 kDa

(12 kDa on Gel) WB – 1:2500 KB Römisch

anti-Sbh1p (rabbit)

Sec61 beta homolog 1 (a.a.

10-23)

9 kDa

(12 kDa on Gel)

WB – 1:2500

IP – 1:200

Pineda Antikörper Service

anti-Sec61p (rabbit)

Sec61 (N-terminus)

52 kDa

(42 kDa on Gel) WB – 1:2500 KB Römisch

anti-Wbp1p (rabbit)

Wheat germ agglutinin-

Binding Protein 49 kDa

WB – 1:2000

IP – 1:200 M. Aebi

pRS415 SBH1 S3A/T5A TMDM

LEU2 / Amp KRB1048 J. Simon

pRS415 SBH1 TMDM LEU2 / Amp KRB1050 J. Simon

37

anti-rabbit (HRP conjugated)

(goat) Rabbit IgG - WB – 1:10000

Rockland™: cat. # 611-1302

1.6 - Softwares

Table 14 - Softwares used in this study

Company Software

Bio-Rad Laboratories, Inc. Quantity One 4.6.2 Basic

National Institute of Health ImageJ

GE Healthcare Image Quant™ TL 8.1

Typhoon Scanner Control

2. METHODS

2.1 - Sterilization

All glassware and media were sterilized by autoclaving at 100 kPa and 120°C for 20 min if not stated otherwise.

2.2 - Growth of S. cerevisiae

S. cerevisiae cells were grown at 30°C in YPD with continuous shaking at 200 rpm or on YPD or drop-out plates at 30°C if not stated otherwise (Table 4)

2.3 - Growth of E. coli

E. coli cells were grown at 37°C, if not stated otherwise, in LB medium with continuous shaking at 220 rpm or on LB medium plates (Table 4).

2.4 - Plasmid Extraction

The kit Invisorb® Spin Plasmid Mini Two (Stratec Molecular, Birkenfeld, Germany) was used for plasmid extraction from bacteria, to later transform yeast cells with it.

To extract the needed plasmid, 2 mL of an E. coli overnight (o.N.) culture were transferred in a micro-tube and centrifuged for 1 min at 13.000 rpm to pellet the cells. After removing the supernatant, the cells were resuspended in 250 µL of Solution A, to weaken the cell walls and then 250 µL of Solution B were added to perform the lysis. Finally, 250 µL of Solution C were added to stop the lysis, and the samples were centrifuged for 5 min at 13.000 rpm. The supernatant was then transferred onto a Spin Filter, incubated for 1 min at room temperature (RT) and centrifuged for 1 min at 13.000 rpm. After the filtrate was discarded, 750 µL of Wash Solution were added and the samples were centrifuged at 13.000 rpm for 1 min, and then centrifuged again at 13.000 rpm for 3

38

min, to remove the residual ethanol (EtOH) from the filter. Finally, 50 µL of hot (~ 65°C) sterile water was added in the middle of the filter, incubated 1 min at RT and centrifuged at 13.000 rpm for 1 min to elute the plasmid.

Plasmid concentration was determined using the NanoDrop 200oc spectrophotometer from Manfred Schmitt’s laboratory.

2.5 - Transformation of S. cerevisiae

Yeast cells were transformed in order to study the influence of given mutations on specific proteins or organelles.

The protocol used for yeast transformation was the Lithium Acetate (LiAc) Transformation protocol. In order to perform the transformation, 2 mL of a S. cerevisiae o.N. culture were harvested at 3.000 rpm for 2 min. The pellet was then washed with 1 mL of LiAc/TE Buffer and centrifuged for 2 min at 3.000 rpm. The pellet was then resuspended in 100 µL of LiAc/TE and 20 µL of denatured Carrier-DNA (salmon sperm), 1 µg of plasmid DNA (pDNA), 600 µL of PEG-Buffer and 50 µL of 10x LiAc were added. The samples were incubated for 1 h at 30°C. After this incubation, 20 µL of DMSO were added, and the samples were incubated for 15 min at 42°C. They were then centrifuged for 2 min at 3.000 rpm and washed two times with 1 mL of 1x TE. Finally the pellet was resuspended in 100 µL of 1x TE and plated on corresponding auxotrophy plates.

2.6 - Preparation of Cell Extracts

Cell extracts were prepared in order to investigate soluble or membrane proteins of the strains employed in this study.

The preparation of whole-cell extracts requires an OD600 between 1 and 2 (when yeast cells are in the exponential growth phase).

The cells were concentrated to 2 OD600/mL. 1 mL was taken and centrifuged at 13.000 rpm for 1 min. They were then resuspended in 1 mL of sterile water and centrifuged at 13.000 rpm for 1 min. The pellet was then resuspended in 100 µL of 200 mM DTT 2x Sample Buffer. Half of the volume of Glass beads was added and the lysis was performed with a Beadbeater (Bio Spec Products Inc., Bartlesville, USA). Two 1 min cycles of disruption were performed with 1 min of incubation at 4°C between each one. The samples were finally denatured for 5 min at 95°C or for 10 min at 65°C if soluble or membrane proteins had to be investigated, respectively.

2.7 - Protein Gel Electrophoresis and Western Blot Analysis

2.7.1 - Protein Gel Electrophoresis

Protein Gel Electrophoresis was employed to resolve proteins according to their molecular weight.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was carried using NuPage® 4-12 % Bis-Tris precast gels or self-made 7,5 % SDS-Gels. 6 µL Page Ruler Prestained Protein Ladder (Thermo Scientific, Waltham, USA) and 20 µL of each sample were loaded. Precast gels were ran in 1x MOPS buffer (for better resolution of high molecular weight proteins) or 1x MES buffer (for better resolution of low molecular weight proteins), self-made SDS-gels were ran in 1x Running Buffer, at 80 V for 15 min and then at 160 V for 1 h.

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2.7.2 - Western Blot Analysis

Western Blotting was employed to identify the desired proteins using appropriate antibodies.

Proteins were transferred onto nitrocellulose membranes. The blot was assembled using the nitrocellulose membrane as well as Whatman® Paper and sponges soaked in Transfer Buffer in a Transfer Cell (Bio-Rad Laboratories, München, Germany) at 4°C for 1 h 30 min at 100 V, or o.N. at 0.3 A.

Following the transfer, the membranes were blocked in Milk Buffer under shaking for 1 h, at RT or o.N. at 4°C. The membranes were then incubated under shaking with the specific antibody (diluted in Milk Buffer), for 2 h at RT or at 4°C o.N.. The membranes were subsequently washed twice in Milk Buffer and twice in TBS-T for 10 min. They were then incubated with the secondary antibody (anti-rabbit, diluted in TBS-T) for 1 h under shaking at RT, and later washed 5 or 6 times

for 5 min with TBST. Finally, the membranes were covered with Detection Reagent 1 Peroxide

Solution and Detection Reagent 2 Luminol Enhancer Solution or Super Signal® West Dura Stable Peroxide Buffer and Super Signal® West Dura Luminol/Enhancer Solution (Thermo Scientific, Waltham, USA) and revealed by enhanced chemoluminescence (ECL) in the CAWOmat 2000 (CAWO Solutions, Schrobenhausen, Germany), ChemiDoc XRS (Bio-Rad Laboratories, München, Germany) or Amersham Imager 600 RGB (GE Healthcare, Freiburg, Germany).

2.7.3 - Membrane stripping

In order to remove primary and secondary antibodies from a WB membrane and reprobe it with a different primary antibody, the membranes were washed in TBS-T for 5 to 10 min, and then incubated in Restore™ Western Blot Stripping Buffer (Thermo Scientific, Waltham, USA) for 15 min at RT. Afterwards, they were washed two times 5 min with TBS-T and the immunoblotting was performed again.

2.8 - Cycloheximide Chase Experiments

Cycloheximide chase was performed to investigate ERAD defects, using a misfolded variant of the protein CPY.

Strains of interest were grown o.N. at 30°C to an OD600 of 1-2. 0,2 mg/mL cycloheximide was added to the liquid cultures. At t=0, 1 mL duplicates, with a concentration of 2 OD600/mL were taken. Regular amount of cells were removed every 15 min for 60 min.

Cell extracts were then prepared as described in 2.6.

2.9 - Pulse Experiments

2.9.1 - Pulse Labeling

The Pulse labeling experiment was employed to identify desired proteins with more specificity than the Western Blot, using radioactive peptides and immunoprecipitation. The labeling mix is composed of 35S-labeled L-methionine (Met) and 35S L-cysteine (Cys). Since the cells are taken while they are quite actively proliferating, because they are in the beginning of the exponential phase, these labeled amino-acids will be assimilated and incorporated during protein synthesis. This causes all new synthesized proteins to be 35S labelled, which enables a very sensible detection of newly synthesized proteins.

40

Strains of interest were grown o.N. in Minimal Medium at 30°C, to an OD600 of 0,5-1,5. The cells were harvested at 4.500 g for 5 min and washed twice with 10 mL Labeling Medium. They were then concentrated to 6 OD600/mL. 250 µL were taken in duplicate from each culture, in order to have an OD600 of 1,5 in each sample. They were then incubated at 30°C for 20 min under shaking (800 rpm). 4,5 µL of Labeling Mix (EXPRE35S35S Protein Labeling Mix, Perkin Elmer) were added to each sample. The labeling was stopped after 5 min using 750 μL of ice-cold Tris-Azide. The samples were then centrifuged for 1 min at 13.000 rpm, and washed again with 1 mL of Tris-Azide. The pellets were resuspended in 1 mL of Resuspension Buffer and incubated at RT for 10min. After a 13.000 rpm 1 min centrifugation, 150 μL of Lysis Buffer and half of the volume of Glass beads were added and the cells were lysed using a Beadbeater. Three 1 min cycles of disruption were performed with 1 min of incubation at RT between each one. Samples were then centrifuged (1 min, 13.000 rpm) and incubated at 95°C for 5 minutes. Finally, the samples were washed three times with 250 µL of IP Buffer w/o SDS and the supernatants were collected.

2.9.2 - Immunoprecipitation

Supernatants collected in the previous step were incubated on a rotary wheel for 30 min at RT with 60 μL of Protein-A Sepharose for a pre-clearing. They were then centrifuged (1 min, 13.000 rpm) and the supernatants were collected. 5 µL to 10 µL of specific antibody were added, as well as 60 μL of Protein-A Sepharose and 1 mL of IP Buffer with SDS. The samples were incubated for 2 h at RT (or overnight at 4°C) on a rotary wheel. After a quick spin (1 min, 13.000 rpm), the supernatants were collected in a new tube and frozen for later immunoprecipitation with different antibodies. Each pellet was washed two times with 1 mL of IP Buffer, once with 1 mL of Urea Wash, 1 mL of ConA Wash, 1 mL of Tris-NaCl. 20 μL of 200 mM DTT 2x Sample Buffer were then added and the samples were heated for 5 min at 95°C.

2.9.3 - Gel Electrophoresis and Revealing

The samples were resolved as described in 2.7.1.

25 µL of each sample and 6-8 µL Page Ruler Prestained Protein Ladder/[Methyl-14C] Methylated Protein Molecular Weight Marker (Perkin Elmer) were loaded

Afterwards, the gel was fixed in Fixing Solution 1 for 15 min and in Fixing Solution 2 for 30 min.

Finally, the gel was dried for 2 h at 80°C in gel dryer (583 Gel Dryer, Bio-Rad Laboratories, München, Germany) and a phosphor plate (GE Healthcare, Freiburg, Germany) was exposed to it for 12-24 h.

2.10 - Native Immunoprecipitation

Native immunoprecipitation experiment was carried to study the interaction between Sbh1p and the OST.

Strains of interest were grown o.N. at 30°C, to an OD600 of 1-2. Cells were concentrated to 2 OD600/mL, and 5 samples of 1 mL each were taken. Samples were centrifuged at 8.000 rpm for 2 min. They were then resuspended in 50 μL of 100 mM Tris-HCl pH 9,4, incubated for 10 min at RT and centrifuged at 8.000 rpm for 2 min.

All the subsequent steps were performed on ice or at 4°C.

41

The samples were resuspended in 100 μL of Lysis Buffer w/o Glycerol, and half of the volume of Glass Beads was added. The cells were lysed using a Beadbeater. Three 1 min cycles of disruption were performed with 1 min of incubation at RT between each one. After a short centrifugation (2.000 rpm for 1 min), all supernatants of the same strain were taken together in a new tube, and centrifuged for 10 min at 13.000 rpm. The pellet was resuspended in 50 µL of Lysis Buffer. 200 µL of Lysis Buffer with 3,75% digitonin were then added, and the samples were incubated on ice for 30 min. After another centrifugation at 13.000 for 10 min, the supernatant was taken in a new tube and brought to 1 mL with Lysis Buffer. The Protein-A Sepharose was washed five to six times in Lysis Buffer to be equilibrated. Then, 60 μL of Protein-A Sepharose were added to the supernatants that were then incubated on a rotary wheel for 30 min at 4°C for pre-clearing. The samples were then centrifuged and the supernatants were taken in a new tube. 5 µL to 10 µL of specific antibody were added, as well as 60 μL of Protein A Sepharose and 1 mL of Lysis Buffer, and incubated for 2-4 h at 4°C on a rotary wheel. After a quick spin (1 min, 13.000 rpm), the pellet was washed five to six times with Lysis Buffer. Finally, 20 µL of 2x Sample Buffer were added, and the samples were heated for 10 min at 65°C.

The samples were then resolved using an SDS-Gel and the proteins were then transferred onto nitrocellulose for immunoblotting (ref. 2.7.2).

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III. Results

1. IMPORT OF GLUCOSIDASE I INTO THE ER

Glucosidase I is an important enzyme that plays a role in the proper folding of glycoproteins. It removes the α-1,2-linked glucose residue from the oligosaccharide chain of glycoproteins, in order to allow a second glucosidase (Gls2) to remove the second glucose residue from the chain. If Gls1p doesn’t remove the first glucose residue, Gls2 won’t have access to the second glucose, the glycoprotein will not be recognized by the ERQC, and be targeted to the ERAD for degradation by the 26S proteasome (ref. I.3.3).

Thus it is important that Gls1 is correctly imported into the ER, and this is what we investigated in the following mutants.

1.1 - Phosphorylation- and transmembrane double mutants

As it was described in the work of Soromani et al. (2012), T5 and S3 are two important phosphorylation sites discovered on the cytosolic domain of Sbh1p. The mutation of Thr to Ala at position 5 results in a destabilization of Sbh1p, but not in a hypophosphorylated protein. This result suggested that the kinase responsible for the phosphorylation of the T5 was able to use other residue as substrate, and the Ser at position 3 was proposed as a candidate. The T5 phosphorylation site on Sbh1p evolved twice independently (in S. cerevisiae and mammals) and is thus suggested to play a particularly important role, even more because it is only phosphorylated when part of the Sec61 complex.

Another domain of Sbh1p has been shown to have an important role: the TMD (Feng et al., 2007). In fact, the expression of the TMD alone neither affect the functionality of the protein nor its interaction with the Sec61 complex. The import of N-glycosylated α-factor precursor (3gpαf) by the Sec61 complex was investigated in Δsbh1Δsbh2 cells in order to detect defects in the functionality of Shb1p due to the lacking of its cytosolic and transmembrane domains. This led to the discovery that Δsbh1Δsbh2 cells show a reduced level of Msn1p due to constitutive translocation defect, further leading to a defect in the N-glycan trimming by glucosidase and mannosidase.

As the role of the TMD wasn’t defined, Zhao and Jäntti (2009) decided to characterize the function of the latter. They thus generated random mutations in this domain, to observe how they influenced the protein translocation. This led to the discovery, that a combination of two point mutations in the TMD (P54S, V57G) results in the inactivation of the transmembrane domain.

The import of Gls1 in T5A, S3A, as well as in the mutant carrying two point mutations in the transmembrane domain (P54S, V57G; Zhao and Jäntti, 2009) was investigated in Δsbh1Δsbh2 cells, in order to identify a defect in the translocation of this enzyme.

On Figure 6, the results of the performed Western Blot are presented for the three mutants mentioned above, as well as strains carrying the wild-type SBH1 gene (WT SBH1), the vector control (pRS415) and the WT strain expressing both Sbh1p and Sbh2p.

The WT strain clearly shows an intense band, indicating that Gls1 was correctly imported. The S3A mutant only displayed a mild defect in the import of Gls1, while a more pronounced defect was shown by the T5A and the transmembrane double mutant (TmDM). The WT SBH1 and pRS415 strains manifest a very strong defect in the import of Gls1.

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Figure 6 - Import of Gls1 in SBH1 mutant strains. Transformants and the corresponding WT were grown at 30°C to an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-PAGE. After transfer on nitrocellulose membrane, they were immunoblotted with anti-Gls1p and anti-Cdc48p (loading control) antibodies. The P54S, V57G strain was only expressing the TMD.

The following graph (Figure 7) represents the average amount of Gls1 in the cell for all strains for three repetitions of the experiment.

Figure 7 - Amount of Gls1 in the cell for different SBH1 mutants. Quantification was performed using ImageJ. Error bars indicate the standard error.

Less Gls1 is present in the mutants compared to the WT, but the strain carrying the S3A mutation seems to have the stronger import of Gls1 even though the error bar indicate that it might not be significant. The empty plasmid also shows a stronger import than the other mutants. The T5A and WT SBH1 strains carry a slightly lower amount of Gls1, whereas the P54S, V57G mutant clearly present a very reduced import of this protein.

In order to have a more precise idea of the behavior of these mutants, a Pulse experiment has been performed. However, the immunoprecipitation was unsuccesful, and no signal could be detected.

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The TmDM used for all experiments only contains the TMD, in p425ADH. The TmDM used for the experiment shown in Figure 8 was generated by Julien Simon in the same plasmid (pRS415) as the other mutants, and expresses the whole Sbh1 protein containing the transmembrane mutations.

As expected, the WT strain shows a robust import of Gls1 in the ER, as do the T5A strain and the TmDM. The WT SBH1 strain also shows a strong band, but a slightly smaller amount of Gls1 was imported. The S3A strain seems to import slightly less Gls1 than the WT SBH1, while a weak band was displayed by the vector control.

Figure 8 - Import of Gls1 in SBH1 mutant strains. Transformants and the corresponding WT were grown at 30°C to an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-PAGE. After transfer on nitrocellulose membrane, they were immunoblotted with anti-Gls1p and anti-Cdc48p (loading control) antibodies.

Thus, we observe a very different behavior between the TmDM expressing the whole Sbh1 protein and the one expressing only the TMD. This mutant was generated at the end of Julien Simon’s Bachelorthesis, I was thus unable to repeat this experiment due to time constraints.

1.2 - Combined mutants

The mutants used for the second part of my Bachelorthesis were generated by Julien Simon

using site-directed mutagenesis. These mutants correspond to combinations of the previously

tested mutations: S3A/T5A; TmDM + S3A; TmDM + T5A, TmDM + S3A/T5A (ref. II.1.3 - Tables

10/11/12).

The Western Blot Analysis of those mutants was performed in order to investigate the effect

of combined mutations on the import of Gls1 into the ER lumen.

The results of this experiments are shown in Figure 9. Since most of the mutations are

combined with the mutations in the TMD, the TmDM expressing the whole Sbh1 protein was used

as a control in addition to the controls used for the previous experiment.

The WT strain does not show any Gls1 import defect; the TmDM and S3A+TmDM are almost comparable to the WT in the import of this protein. A slightly reduced import into the ER is observed in the T5A+TmDM, S3A/T5A+TmDM and the WT SBH1 strains. The intensity of the band in the S3A/T5A and pRS415 strains are very low, indicating a strong import defect of Gls1.

In order to have a better understanding of the results, the bands were normalized against

the corresponding loading control. The mean results for both duplicates of this experiment are

shown in Figure 10.

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Figure 9 - Import of Gls1 in SBH1 combined mutant strains. Transformants and the corresponding WT were grown at 30°C to an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-PAGE. After transfer on nitrocellulose membrane, they were immunoblotted with anti-Gls1p and anti-Cdc48p (loading control) antibodies.

Figure 10 - Amount of Gls1 in the cell for different combined SBH1 mutants. Quantification was performed using ImageJ. Error bars indicate the standard error.

Less Gls1 is expressed in the mutants compared to the WT, but the strain carrying the S3A/T5A+TmDM mutation seems to have a stronger import of Gls1 than the other mutants. The WT SBH1 strain also seems to import more Gls1 than the other mutants, which is consistent with the expression of the WT protein. The S3A+TmDM and T5A+TmDM strains show a similar import of Gls1, and the amount imported by the TmDM is also comparable. The empty plasmid strain and the S3A/T5A mutant, however, manifest a strong defect in the import of Gls1, compared to the other mutants and to the WT.

The quantification of this experiment shows large error bars. In order to have a more precise idea of the behavior of these mutants, the experiment has to be repeated. Julien Simon managed to generate two of those mutants only in the penultimate week of our Bachelorthesis. Due to time constraints, I was unable to perform more repetitions. For the same reason, a Pulse experiment of these mutants could not be conducted.

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2. OLIGOSACCHARYL TRANSFERASE COMPLEX AND SBH1P

Several forms of CPY can be found in the cell. There is a proCPY (pCPY) polypeptide of 59 kDa found in the cytosol, an ER form (p1CPY) of 67 kDa, a Golgi form (p2CPY) of 69 kDa, and finally the mature form (mCPY) of 61 kDa. Their molecular weight changes when they are unglycosylated: p2CPY is then 59 kDa, as well as p1CPY, and mCPY is 51 kDa (Stevens et al., 1982).

CPYp was used as a loading control for the immunoblotting of Gls1, but we noticed that for two of the strains, several forms of CPY were present in the cell (Figure 11), so it couldn’t be further used as loading control, as it seemed that the mutations affected its import. That is the reason why we used Cdc48p as a loading control afterwards.

Figure 11 - Import of CPY in SBH1 mutants. Transformants and the corresponding WT were grown at 30°C to an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-PAGE. After transfer on nitrocellulose membrane, they were immunoblotted with anti-Gls1p and anti-Cdc48p (loading control) antibodies. The P54S, V57G strain was expressing the whole SBH1 protein containing those mutations in the TMD.

For every strain, at least two bands are detected, obviously corresponding to p2CPY (69 kDa) and p1CPY (67 kDa). Nevertheless, there are two more bands detected for the TmDM and the pRS415 strain. They probably correspond to the mCPY (61 kDa), the pCPY (59 kDa) and the unglycosylated forms of p1CPY and p2CPY (both 59 kDa).

This led us to think that the double mutation in the transmembrane domain of Sbh1p might

lower the interaction of the latter with the OST, thus resulting in an underglycosylation of this

protein. In order to test this hypothesis, a native IP of the Sec61 complex interacting with the OST

has been performed.

The native IP (ref. II.2.10) was first performed using antibodies directed against Wbp1p on

the TmDM and the WT strain. Anti-Wbp1p and anti-Sbh1p antibodies were then used for the

immunoblotting. Unfortunately, we couldn’t detect any Sbh1p in neither of the strains, and we were

only able to detect the heavy chain (HC) of the antibody (not shown).

This experiment was repeated using monospecific anti-Sbh1p antibodies for the IP instead

of anti-Wbp1p. The immunoblotting was performed using anti-Wbp1p and monospecific anti-Sbh1p

antibodies. The same results as in the first experiment were obtained: we could only detect the HC

of the antibody (not shown).

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3. ER-ASSOCIATED DEGRADATION DEFECTS

When proteins are not able to reach their native conformation, they are degraded by the 26S proteasome in the cytosol following the ERAD pathway. This prevents the accumulation of toxic substrates in the ER, which can lead to cell death (ref. I.3.4).

Sometimes, this degradation pathway doesn’t work properly, and there is an accumulation of misfolded proteins in the lumen of the ER. We investigated the ERAD of several mutant strains, following the degradation of a mutant form of CPY, named CPY*.

3.1 - Phosphorylation- and transmembrane double mutants

In order to identify ERAD defects in the mutant strains we generated, cycloheximide chase experiments were performed (ref. II.2.8). The cycloheximide blocks the translation of new mRNAs, thus allowing us to follow the destiny of the already translated and translocated proteins. The mutant variant of CPY (G225R; allele: prc1-1), CPY*, is recognized by the ERQC, retrotranslocated in the cytosol and degraded by the 26S proteasome (Finger et al., 1993).

Strains containing the mutations of interest (S3A, T5A and the transmembrane double mutation), as well as three controls (WT SBH1, pRS415 and Δder1) were first transformed with the plasmid pDN431 carrying the gene encoding the mutated variant of CPY, tagged with Hemagglutinin (HA).

The results of one of these experiments are shown in Figure 12. It has been previously demonstrated that a mutation in DER1 results in an ERAD defect, thus the Δder1 mutant was used as a positive control (Knop et al., 1996).

After 15 min of chase, half of the CPY* carried by the WT SBH1 strain (red line) was already degraded; in the S3A (purple line) and T5A (light blue line) mutants the half-life of CPY* was extended to 20 min. Nevertheless, about 90% of the CPY* in those cells was degraded after 45 min, and 95 to 97 % after 60 min. These data indicate that those mutants do not show any ERAD defect.

After only 15 min, half of the CPY* was degraded in the Δder1 (orange line) cells but afterwards it wasn’t degraded anymore as the amount stays relatively identical. After 60 min of chase, only 60% of CPY* was degraded. This final amount of CPY* is almost the same for the TmDM (dark blue line), although it was degraded slower than in the Δder1 cells. Indeed, the degradation of half of the CPY* present in the cell needed about 50 min. The degradation of this substrate for the strain carrying the empty plasmid pRS415 (green line) was very slow and only 30% of the CPY* was degraded at the end of the experiment. Those three strains, including the positive control, show a strong ERAD defect, particularly the pRS415 strain.

The mutants tested showed a comparable behavior in every repetition of this experiment: a strong ERAD defect for the TmDM, pRS415 and Δder1 cells, and a correct degradation for the S3A, the T5A and the WT strains. However, the quantification in each of the assays resulted in a great variability of the quantification values, when expressed as mean values. For this reason, the most representative assays is presented in Figure 12. Another experiment is shown in Figure 15 (Annex VIII.2).

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Figure 12 - Degradation of CPY* in SBH1 mutant strains over time. (A) Cells were grown at 30°C to an OD600=2 in YPD medium. Cycloheximide was added to inhibit translation, and samples were taken every 15 min for 60 min. Whole-cell extracts were prepared and proteins were resolved on SDS-PAGE. They were transferred onto nitrocellulose membranes and immunoblotted with CPY and Rpn12 as a loading control. (B) B) Quantification of the experiment in (A). The analysis was performed using ImageJ.

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3.2 - Combined mutants

In order to identify ERAD defects in the mutant strains (S3A/T5A; S3A+TmDM; T5A+TmDM and S3A/T5A+TmDM) generated by Julien Simon, cycloheximide chase experiments were performed as described in II.2.8. These strains (ref. II.1.3) were transformed with the plasmid pDN431 carrying the gene encoding CPY*HA .

The results of this experiment are shown in Figure 14. A Δder1 mutant was used as a positive control. This experiment could only be performed once due to time constraints.

Only 5 min after the beginning of the chase, half of the CPY* present in the S3A/T5A mutant (blue line) and the S3A+TmDM (green line) was degraded. After 15 min, the amount of CPY* in the S3A/T5A mutant started to increase, and finally reached a concentration of 20% at the end of the chase, whereas the S3A+TmDM already reached its final degradation rate (80%) after 15 min and stayed relatively constant until the end of the chase. The two strains with the higher CPY* degradation rates are the T5A+TmDM (purple line) and the WT SBH1 (orange line) strains, with 85% and 95% of final degradation of CPY*, respectively. The T5A+TmDM showed a half life of CPY* present of about 13 min, and the WT strain of 25 min. The latter degraded CPY* until it reached its maximum rate after 45 min. The T5A+TmDM strain also degraded CPY* constantly until the end of the chase. Those four strains, S3A/T5A, S3A+TmDM, T5A+TmDM and WT SBH1, did not show an ERAD defect.

The first part of the graph is irregular for the following mutants, thus it will need more assays to define it correctly. The TmDM (red line) reached a final CPY* degradation rate of 50% at the end of the chase. The S3A/T5A+TmDM (light blue line) reached almost the same degradation rate, but showed a small accumulation (10%) after 15 min of chase. 70% of the CPY* present at that time was then degraded in 15 min and subsequently the rate stayed relatively stable. After 15 min of chase, the pRS415 (dark blue line) transformant shows a very strong increase of CPY* (about 30%), probably corresponding to a delay in the translocation of that protein into the lumen of the ER. During the following 45 min of the chase, CPY* was degraded, and a final amount of 70% of CPY* was remaining in the cell. In the Δder1 cells (burgundy-colored line), 20% of the present CPY* was degraded after 15 min, and the final degradation rate reached 30%. These four mutant strains, S3A/T5A+TmDM, TmDM, pRS415 and Δder1 showed a strong ERAD defect, particularly the pRS415 strain and the Δder1 cells.

In order to confirm the previously obtained results, this experiment should be repeated.

4. TEST ON ANTIBODIES DIRECTED AGAINST SBH1P

Soromani et al. (2012) investigated the possible influence of phosphorylation of the cytosolic domain of Sbh1p on its growth and on the translocation efficiency. Therefore, they performed site-directed mutagenesis to mutate the presumable phosphorylation site to an unphosphorylable amino-acid (Ala) and in particular, they investigated the phosphorylation on T5 position. Indeed, it seems that this site and the two Pro residues that flank it have evolved independently in S. cerevisiae and in mammals, and they are not present on Sbh2p.

To characterize this mutant (T5A), they performed a western blot, using Sbh1p antibodies raised against the first 18 a.a. of the cytosolic domain of Sbh1p. The signal detected on the membrane for the T5A mutant was reduced compare to the WT.

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Figure 13 - Degradation of CPY* in combined SBH1 mutant strains over time. (A) Cells were grown at 30°C to an OD600=2 in YPD medium. Cycloheximide was added to inhibit translation, and samples were taken every 15 min for 60 min. Whole-cell extracts were prepared and proteins were resolved on SDS-PAGE. They were transferred onto nitrocellulose membranes and immunoblotted with antibodies directed against CPYp and Rpn12p as a loading control. (B) Quantification of the experiment in (A). The analysis was performed using ImageJ.

In order to verify the reliability of the antibody used, they performed another immunoblotting using mutants lacking either SBH1 or SBH2. It was thus shown, that this antibody also recognizes Sbh2p, even though it better recognizes Sbh1p. The only region that is different between both is comprised

51

between the second and the fifth a.a.. As the mutation tested is on the fifth a.a., it is possible that it affected the interaction with the antibody, thus explaining the reduced signals.

A new Sbh1p antibody was raised against the a.a. 10 to 23 and a western blot was performed on following strains in order to possibly confirm the previously formulated hypothesis. The WT (SBH1SBH2) and the T5A mutated strain were used to verify if the latter also shows a reduced signal if immunoblotted with another antibody. Δsbh1SBH2, SBH1Δsbh2 and Δsbh1Δsbh2 were used in order to see if this newly raised antibody also recognizes Sbh2p.

The results obtained after immunoblotting with both antibodies (Sbh1 N-ter and Sbh1 10-23) are presented in Figure 15. The WT strain was used as a positive and Δsbh1Δsbh2 as a negative control. Figure 15 shows the results obtained with the antibody raised against the a.a. 1 to 18 (Sbh1 N-ter; A) and the results obtained using the antibody Sbh1 10-23 (B).

Figure 14 -Test on a new Sbh1p antibody and comparison with the previously used one. Cells were grown at 30°C to an OD600=2, cell-extracts were prepared and proteins resolved on SDS-PAGE. After transfer on nitrocellulose membrane, they were immunoblotted with both anti-Sbh1p antibodies. Anti-Sec61p directed against its N-terminus (Sec61 N-ter) was used as loading control.

In (A), the bands detected for the WT and the T5A mutant strains are comparable to the results described by Soromani et al. (2012): the T5A mutant shows a reduced signal compared to the WT strain. The comparison between the very weak band detected in the Δsbh1SBH2 mutant and the stronger one detected in the SBH1Δsbh2 mutant indicates that the antibody recognizes SBH1 better than SBH2, thereby confirming the results obtained by Soromani et al.

In (B), an intense signal was detected for the T5A mutant, almost as strong as the one detected for the WT. The figure also shows an intense signal for the SBH1Δsbh2 mutant, and a weak one for Δsbh1SBH2.

After comparison with the results obtained by Soromani et al. (2012), the antibody directed against the a.a. 10 to 23 seems to recognize the T5A mutant better than the antibody directed against the N-terminus, and also to better detect Sbh1p. However, both antibodies detect a relatively identical amount of Sbh2p, indicating that the detection for Sbh1p would be comparable for both antibodies.

52

IV. Discussion

1. IMPORT OF GLUCOSIDASE I INTO THE ER

Soromani et al. (2012) discovered that T5 is an important phosphorylation site on Sbh1p but

if mutated to an unphosphorylable a.a. it doesn’t result in a hypophosphorylated protein, thus

suggesting that another site can be phosphorylated by the same kinase, such as the Ser at position

3. Another study, by Feng et al. (2007), demonstrated that a Sbh1 protein expressing only the TMD

was still functionally competent. Moreover they discovered that Δsbh1Δsbh2 cells presented a defect

in the translocation of Msn1, an enzyme that plays a role in the ERQC. Subsequently, in order to

characterize the function of the TMD, Zhao and Jäntti (2009) mutated randomly several a.a. in the

latter, leading to the discovery that the combination of two point mutations (P54S, V57G) resulted

in the inactivation of the TMD.

The aim of the previously described experiment was to investigate the import of Gls1,

another enzyme from the ERQC, in Δsbh1Δsbh2 cells carrying the phosphorylation mutations

mentioned above, and in a strain carrying the two point mutations in the TMD.

Almost 50% less Gls1 was imported in the strain carrying the WT SBH1 than in the WT strain,

but this can be easily explained by the fact that the WT strains carries both SBH1 and SBH2, whereas

the WT SBH1 was transformed in a Δsbh1Δsbh2 strains, and the cells are thus only expressing Sbh1p.

As it has been demonstrated that Sbh2p is part of the Ssh1 complex, that import proteins co-

translationally (Soromani et al., 2012), as does the Sec61 complex, when the gene encoding this

protein is deleted, it is likely that less proteins will be imported co-translationally.

Due to large variation of the Gls1 amount present in the cell depending on the experiments,

the results obtained for the S3A mutant are not significant, but the data indicate that an amount

comparable to the one manifested by the WT SBH1, was imported. Thus, it is possible to say that

this mutant doesn’t have a defect in the import of Gls1 in the ER. The same can be concluded for the

T5A mutant that shows a similar rate of Gls1 import.

The TmDM shows a stronger defect compared to the WT SBH1 control (about 50% less

import) and an even stronger one in regard to the WT strain (around 66%). The small error bar

indicates that for all performed experiments, the behavior is constant. This mutant thus presents a

defect in the import of Gls1 in the ER.

The TmDM used for almost all experiments was lacking both cytosolic and lumenal domains

of the protein and only expressed the TMD. Julien Simon generated a TmDM expressing the whole

Sbh1 protein, and it was used for the last experiment with these three mutants. The latter showed a

band for the TmDM, barely less intense than the WT strain. The results of only one experiment

cannot be very representative, nonetheless it might be possible that the mutations in the TMD of

the full protein do not have any impact on the import of Gls1 in the ER. This would mean that the

deletion of the cytosolic domain, however, induces import defects.

53

The hypothesis previously made is consistent with the data produced by the investigation

of Gls1 import defect by the combined mutants. The TmDM used for the second experiment

expresses the full Sbh1 protein.

In comparison to the WT and the WT SBH1 strains, the mutant carrying the combination of

both S3A and T5A mutations shows a really strong defect in the import of Gls1: about 66% less than

the WT SBH1 strain and 75% less than the WT strain. A mutation from Ser and Thr to alanine (Ala)

prevents the phosphorylation of the corresponding site. Other phosphorylation sites have been

investigated (Soromani et al., 2012) but S3 and T5 positions seemed to be the major ones. It is then

likely that due to insufficient phosphorylation of the protein, the efficiency of translocation is

reduced, which would explain the lower amount of Gls1 detected in this mutant. Moreover, even

though the results weren’t quantified, the TmDM lacking the cytosolic domain (and the lumenal

domain) seemed to show a similarly reduced import, supporting the idea that this defect is somehow

related with the phosphorylation of the cytosolic domain of Sbh1p.

The strains carrying phosphorylation site mutations coupled with the TmDM didn’t show a

defect in the import of Gls1. However, it seems that the amount of Gls1 present in the cell, which is

similar for both S3A+TmDM and T5A+TmDM mutants, is stronger in the latter than in the TmDM

alone. Otherwise, the amount of Gls1 in the S3A/T5A+TmDM strain is about 1,5 times superior than

the one in the TmDM alone.

Cytosolic domains as well as the TMDs interacts with several protein and complexes. It is

imaginable that a protein or a protein complex might bind to the TMD of Sbh1p and that this

binding allows the interaction of the protein with the cytosolic domain. Mutations in the TMD could

prevent the binding of this protein to the Sbh1 subunit, resulting in a lower interaction with the

cytosolic domain, thus leaving the access free to other phosphorylation sites, improving the

efficiency of the translocation. This theory would explain why more Gls1 was imported in the

mutants combined with the TmDM than in the TmDM alone.

In order to get a better idea of the behavior of these mutants towards the import of Gls1, the

experiment should be repeated. Performing Pulse experiments could also help having more precise

results.

2. OLIGOSACCHARYL TRANSFERASE COMPLEX AND SBH1

The detection of several forms of CPYp in the TmDM and the strain carrying the empty plasmid was interpreted as a defect in the N-linked glycosylation. This hypothesis made sense as CPY has four glycosylation sites, and the molecular weights of the detected forms correlate with the one of the unglycosylated CPY forms (Stevens et al. 1982).

We attributed this defect to a lowered interaction between the OST and Sbh1p, due to the mutations in the TMD. The native IP of both Sec61 complex and OST from the TmDM and the WT strain should have confirmed this supposition.

The IP was performed using anti-Wbp1 antibodies, with Wbp1p being a subunit of the OST, and the second one with anti-Sbh1 antibodies, but none of them gave satisfactory results. After

54

immunoblotting, only the HC of the antibodies was detectable. It was very unlikely that this was due to the antibodies used, as the two experiments were performed using different antibodies.

This defect in the interaction with the OST could be an explanation for the detected bands of the mutant carrying the empty plasmid. Indeed one of the OST subunits, OSt3p or OSt6p, interacts with either Sbh1p or Sbh2p. As the strain carrying the empty plasmid is lacking both Sbh1p and Sbh2p, it is possible that the OST is not able to interact with the translocation complex, thus explaining the N-linked glycosylation defect. However, it has been demonstrated that the OST directly binds to the 60S ribosomal subunit (Harada et al., 2009).

We realized after those two unsuccessful experiments that the TmDM used was only expressing the TMD, thus explaining why no signal could be detected for Sbh1p in neither of the IPs. At the end of our Bachelorthesis, Julien Simon generated the TmDM expressing the whole Sbh1 protein, however, this mutant didn’t show the several CPY forms that were expected (Figure 16, Annex VIII.2), and the same behavior was detected for all combined TmDM generated by Julien (Figure 17, Annex VIII.2).

We thus concluded that the mutations in the TMD does not affect the interaction with the OST, and therefore no glycosylation defect is observed, but the lack of cytosolic or lumenal domains might affect the interaction between OST and Sbh1p.

Nevertheless it is more likely that the detection of those several CPYp forms are related with

a delay in the translocation of CPY. If there is a post-translational import defect, it is difficult to

discern between a delayed translocation and an accumulation of the protein.

3. ER-ASSOCIATED DEGRADATION DEFECTS

Misfolded proteins are toxic for the cell, which is the reason why as soon as they are

recognized by the ERQC, they are directed to the cytosol by the ERAD pathway to be degraded by

the 26S proteasome (Römisch, 2004). When the function of ERAD is compromised, those toxic

substrates accumulate in the lumen of the ER, as they can not be retro-translocated for degradation.

This situation is associated to severe illnesses (more than 60 human diseases have been linked to

the ERAD Pathway), thus it is important to control the proper functioning of this pathway

(Guerriero and Brodsky, 2012).

Mutations in proteins of the complexes that regulate the retrograde transport of proteins to

the cytosol can compromise the ERAD, and since the Sec61 translocon is part of one those

complexes, mutations in its subunits have been demonstrated to lead to severe ERAD defects.

Although it has been show that Sbh1p and Sbh2p are dispensable for ERAD under specific conditions

in vitro, this has not been demonstrated whether this was also the case in vivo (Feng et al., 2007).

Therefore, we performed cycloheximide chase on several mutants, carrying mutations at

phosphorylation sites and/or in the TMD, to follow the degradation of a mutant protein that

undergoes the ERAD pathway, in order to identify defects in this process.

The first mutations investigated were the mutation of two phosphorylation sites of Sbh1 (S3A

and T5A) identified by Soromani et al. (2012) and a mutant carrying two mutations in the TMD

(P54S, V57G) identified by Zhao and Jäntti (2009).

55

The mutations in the phosphorylation sites did not affect the ERAD, as these mutants

efficiently degraded CPY*. T5 is a major phosphorylation site in the Sbh1 cytosolic domain, and it

was proposed that S3 might be too, but other phosphorylation sites were predicted using

bioinformatics tools (Soromani et al., 2012). Even though S3 and T5 sites are not phosphorylable

anymore, the phosphorylation might occur on a different site, thus explaining why these mutants

did not manifest any influence on ERAD.

However, ERAD defects where identified for the TmDM and the pRS415 strain. This TmDM

was only expressing the TMD, suggesting that this defect might be due to the lack of the cytosolic

domain. As the pRS415 strain does not express Sbh1 at all, it is likely that this protein has a specific

function in the ERAD, since a very strong defect is observed in its absence.

It can thus be proposed that Sbh1p might play a major role in the ERAD in vivo, as its

complete deletion leads to very strong defects in degradation of misfolded CPY. Moreover, the

deletion of both its cytosolic and lumenal domains in the TmDM led to a CPY* degradation defect,

suggesting that one or both of them is also important for the retrotranslocation of proteins into the

cytosol. It was previously proposed that this subunit enhances the speed of tranlocation (Soromani

et al. 2012), and the data obtained in this study suggest that it is likely that Sbh1p might be needed

for normal retrotranslocation rates, explaining the accumulation of misfolded CPY in the mutant

strain lacking SBH1.

The second experiment was performed using mutants carrying combinations of the

mutations previously investigated.

The S3A/T5A mutant, S3A+TmDM and T5A+TmDM showed rapid degradation of CPY*,

indicating that ERAD was not affected. However, in addition to the pRS415 strain, strong ERAD

defects were detected in two other strains: the TmDM expressing the whole Sbh1 protein and the

S3A/T5A+TmDM.

The defect observed for S3A/T5A+TmDM suggests that the cytosolic domain of Sbh1p plays

an important role in the retrotranslocation of misfolded proteins. This indicates that the lack of

these two major phosphorylation sites affects the efficiency of protein degradation. Thus, it can be

proposed that the phosphorylation of the cytosolic domain of Sbh1p might be needed to enhance

the efficiency of retro-translocation. This experiment also showed, that point mutations in the TMD,

induce a strong accumulation of misfolded substrates. These data confirm, as it was suggested

previously, that Sbh1p plays a major role in the ERAD pathway.

In conclusion, we have been able to show that cells not expressing Sbh1p are defective in the

retrotranslocation of proteins into the cytosol for degradation, and cells carrying mutations in the

TMD, in the cytosolic domain and lacking the latter manifest a strong accumulation of misfolded

proteins.

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4. TEST ON ANTIBODIES DIRECTED AGAINST SBH1P

The observation of a reduced signal in a SBH1 mutant used by Soromani et al. (2012) was attributed to the possible impaired interaction between the antibody used and the protein, due to the localization of the investigated mutation. In order to test this hypothesis, a commercial anti-Sbh1p antibody, that recognizes an epitope close to the TMD (a.a. 10-23), was tested on the same mutants as Soromani et al.

The comparison between the results obtained with the new antibody and the ones obtained with the antibody directed against the N-terminus of the protein indicates that the antibody raised against a portion closer to the TMD recognizes better Sbh1p than the other one, although it also recognizes Sbh2p, as it was already observed for the N-ter anti-Sbh1 antibody.

Moreover, the affinity of the new antibody seems to be the same for both the WT and the T5A strain. This confirmed the hypothesis formulated by Soromani et al.: the reduced signal observed for the T5A mutant is due to a lowered interaction with the antibody used, as it seems that the region containing this mutation is decisive for antibody binding.

It can thus be concluded that, in the case the N-terminus of Sbh1p has been altered in any way, the use of the antibody raised against a.a. 10-23 is preferable to the use of the one raised against the first 18 a.a., in order to better detect the protein.

57

V. Conclusion Proteins can be translocated in the ER co- or post-translationally, in order to reach their conformation and be led to their final destination. In eukaryotic cells, the major translocation complex is called the Sec61 complex and consist of three subunits, named Sec61p, Sbh1p and Sss1p in yeast. After their translocation, the proteins may suffer several post-translational modification, such as N-linked glycosylation or formation of disulfide bonds. Those modifications contribute for the correct folding of the proteins that can be further directed to the Golgi in transport vesicles so they can be further modified and released where needed. Due to several biological, chemical or physical reasons, it sometimes happens that the proteins are not properly folded. In order to prevent their accumulation in the ER lumen, a specific process called ERAD mediates their retrotranslocation in the cytosol for degradation. However, this process can also fail, thus leaving the substrates to aggregate, resulting in a very high toxicity for the cell.

This study investigates the influence of point mutations in the Sbh1 subunit of the translocon mentioned previously on the translocation of a specific protein, Glucosidase I, in the ER and on the retrotranslocation of misfolded proteins in the cytosol. Two of the mutations studied are localized at phosphorylation sites: one of them has been shown to destabilize Sbh1p, while the other was a suggested to be phosphorylated by the same kinase. It has also been demonstrated that the TMD of Sbh1p alone is sufficient to ensure its function, and that two mutations (P54S, V57G) in the latter inactivate the domain.

The data that resulted from the experiments indicated that the mutations of two phosphorylation sites at the same time led to translocation defect, as well as the deletion of the cytosolic domain of the protein and the mutations in the TMD. On the contrary, mutations in the phosphorylation sites combined to the mutations in the TMD manifested a stronger import of the studied protein. This results suggest that the cytosolic domain of Sbh1p plays an important role in the translocation of proteins, which could go further than just the speed enhancement role that it was attributed to the subunit.

The loading control used for the first experiment presented irregularities that led to the hypothesis that the mutant carrying the mutations in the TMD could present glycosylation defects. However, the investigations didn’t led to that conclusion, and it seems more likely that the several forms of the protein detected were due to a delay in the translocation.

The study of the ERAD pathway in those mutants led to interesting results, as a defect was identified in several of them. These defects pointed out that the cytosolic domain is also important for retrotranslocation, as does the TMD. One of the control also indicated that even though Sbh1p is not essential for the cell’s survival, it deletion leads to severe ERAD defects.

Although experiments have to be repeated to confirm the results, all these data suggest that Sbh1p might play a bigger role in the translocon than what was stated about it. It’s a protein that needs to be further investigated in order to completely identify its function in the translocation, as well as in the retrotranslocation.

58

VI. References

Aebi, M. (2013) N-linked protein glycosylation in the ER. Biochim Biophys Acta 1833(11): 2430-2437.

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2002) Molecular Biology of the Cell, 4th edition. New York: Garland Science. 1616 p.

Berg, J.M., Tymoczko, J.L., Stryer, L. (2002) Biochemistry, 5th edition. W. H. Freeman. 1100p.

Blobel, G., Dobberstein, B. (1975) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 67(3): 835-851.

Borgese, N., Fasana, E. (2011) Targeting pathways of C-tail-anchored proteins. Biochim Biophys Acta - Biomembranes 1808(3): 937-946.

Braakman, I., Hebert, D.N. (2013) Protein folding in the endoplasmic reticulum. Cold Spring Harb Perspect Biol 5(5): a013201.

Buchberger, A. (2014) ERQC autophagy: Yet another way to die. Mol Cell 54(1): 3-4.

Chavan, M., Lennarz, W. (2006) The molecular basis of coupling of translocation and N-glycosylation. Trends Biochem Sci 31(1): 17-20.

Chavan, M., Yan, A., Lennarz, W.J. (2005) Subunits of the translocon interact with components of the oligosaccharyl transferase complex. J Biol Chem 280(24): 22917-22924.

Cooper, G.M., Hausman, R.E. (2006) The Cell: A molecular approach, 4th edition. Sinauer Associates, Inc. 745p.

Cox, J.S., Walter, P. (1996) A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87(3): 391-404.

Deshaies, R.J., Sanders, S.L., Feldheim, D.A., Schekman, R. (1991) Assembly of yeast Sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex. Nature 349(6312): 806-808.

Ellgaard, L., Helenius, A. (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4(3): 181-191.

Falcone, D., Henderson, M.P., Nieuwland, H., Coughlan, C.M., Brodsky, J.L., Andrews, D.W. (2011) Stability and function of the Sec61 translocation complex depends on the Sss1p tail-anchor sequence. Biochem J 436(2): 291-303.

Feng, D., Zhao, X., Soromani, C., Toikkanen, J., Römisch, K., Vembar, S.S., Brodsky, J.L., Keränen, S., Jäntti, J. (2007) The transmembrane domain is sufficient for Sbh1p function, its association with the Sec61 complex, and interaction with Rtn1p. J Biol Chem 282(42): 30618-30628.

Finger, A., Knop, M., Wolf, D. H. (1993) Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. Eur J Biochem 218(2): 565-574.

59

Finke, K., Plath, K., Panzner, S., Prehn, S., Rapoport, T.A., Hartmann, E., Sommer, T. (1996) A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae. EMBO J 15(7): 1482-1494.

Foresti, O., Ruggiano, A., Hannibal-Bach, H.K., Ejsing, C.S., Carvalho, P. (2013) Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4. Elife 2: e00953.

Gogala, M., Becker, T., Beatrix, B., Armache, J.P., Barrio-Garcia, C., Berninghausen, O., Beckmann, R. (2014) Structures of the Sec61 complex engaged in nascent peptide translocation or membrane insertion. Nature 506(7486): 107-110.

Gruss, O.J., Feick, P., Frank, R., Dobberstein, B. (1999) Phosphorylation of components of the ER translocation site. Eur J Biochem 260: 785-793.

Guerriero, C.J., Brodsky, J.L. (2012) The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology. Physiol Rev 92(2): 537-576.

Hammond, C., Helenius, A. (1995) Quality control in the secretory pathway. Curr Opin Cell Biol 7(4): 523-529.

Hampton, R.Y. (2002) ER-associated degradation in protein quality control and cellular regulation. Curr Opin Cell Biol 14(4): 476-482.

Harada, Y., Li, H., Li, H., Lennarz, W.J. (2009) Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site. Proc Natl Acad Sci U S A 106(17): 6945-6949.

Harada, Y., Li, H., Wall, J.S., Li, H., Lennarz, W.J. (2011) Structural studies and the assembly of the heptameric post-translational translocon complex. J Biol Chem 286(4): 2956-2965.

Heider, M.R., Munson, M. (2012) Exorcising the exocyst complex. Traffic 13(7): 898-907.

Herscovics, A., Orlean, P. (1993) Glycoprotein biosynthesis in yeast. FASEB J 7(6): 540-550.

Hutagalung, A.H., Novick, P.J. (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91(1): 119-149.

Junne, T., Kocik, L., Spiess, M. (2010) The hydrophobic core of the Sec61 translocon defines the hydrophobicity threshold for membrane integration. Mol Biol Cell 21(10): 1662-1670.

Kalies, K., Görlich, D., Rapoport, T.A. (1994) Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex. J Cell Biol 126(4): 925-934.

Kinch, A., Saier, H., Grishin, N.V. (2002) Sec61β – a component of the archaeal protein secretory system. Trends Biochem Sci 27(4): 170-171.

Knop, M., Finger, A., Braun, T., Hellmuth, K., Wolf, D.H. (1996) Der1 a novel protein specifically required for endoplasmic reticulum degradation in yeast. EBMO Journal 15(4): 753-763

Korennykh, A., Walter, P. (2012) Structural basis of the unfolded protein response. Annu Rev Cell Dev Biol 28: 251-277.

Lipschutz, J.H., Lingappa, V.R., Mostov, K.E. (2003) The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum. J Biol Chem 278(23): 20954-20960.

60

Lipschutz, J.H., Mostov, K.E. (2002) Exocytosis: the many masters of the exocyst. Curr Biol 12(6): 212-214.

Lodish, H., Darnell, J.E., Berk, A., Zipursky, L., Matsudaira, P., Baltimore, D. (1999) Molecular Cell Biology 4th edition. W.H.Freeman & Co Ltd. 36 p.

Lord, C., Ferro-Novick, S., Miller, E.A. (2013) The highly conserved COPII coat complex sorts cargo from the endoplasmic reticulum and targets it to the golgi. Cold Spring Harb Perspect Biol 5(2): a013367

Meusser, B., Hirsch, C., Jarosch, E., Sommer, T. (2005) ERAD: the long road to destruction. Nat Cell Biol 7(8): 766-772.

Miller, S., Krijnse-Locker, J. (2008) Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol 6(5): 363-374.

Mothes, W., Prehn, S., Rapoport, T.A. (1994) Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane. EMBO J 13(17): 3973-3982.

Osbourne, A.R., Rapoport, T.A., van der Berg, B. (2005) Protein translocation by the Sec61-SecY channel. Annu Rev Cell Dev Biol 21: 529-550.

Pickart, C.M. (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70: 503-533.

Pilon, M., Römisch, K., Quach, D., Scheckman, R. (1998) Sec61p serves multiple roles in secertory precursor binding and translocon into the endoplasmic reticulum membrane. Mol Biol Cell 9: 3455-3473.

Rapoport, T.A. (1992) Transport of proteins across the endoplasmic reticulum membrane. Science 258: 931-936.

Römisch, K. (2004) A cure for traffic jams: small molecule chaperones in the endoplasmic reticulum. Traffic 5(11): 815-820.

Römisch, K. (2005) Endoplasmic Reticulum-Associated Degradation. Annu Rev Cell Dev Biol 21: 435-456.

Ruggiano, A., Foresti, O., Carvalho, P. (2014) Quality control: ER-associated degradation: protein quality control and beyond. J Cell Biol 204(6): 869-879.

Schubert, U., Anton, L.C., Gibbs, J., Norbury, C.C., Yewdell, J.W., Bennink, J.R. (2000) Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404(6779): 770-774.

Schuldiner, M., Metz, J., Schmid, V., Denic, V., Rakwalska, M., Schmitt, H.D., Schwappach, B., Weissman, J.S. (2008) The GET complex mediates insertion of tail-anchored proteins into the ER membrane. Cell 134(4): 634-645.

Silberstein, S., Gilmore, R. (1996) Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase. FASEB J 10(8): 849-858.

Soromani, C., Zeng, N., Hollenmeyer, K., Heinzle, E., Klein, M.C., Tretter, T., Seaman, M.N., Römisch, K. (2012) N-acetylation and phosphorylation of Sec complex subunits in the ER membrane. BMC Biology 13: 34.

61

Stevens, T., Esmon, B., Schekman, R. (1982) Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell 30(2): 439-448

Tabas, I., Ron, D. (2011) Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol 13(3): 184-190.

Toikkanen, J.H., Miller, K.J., Soderlund, H., Jantti, J., Keranen, S. (2003) The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae. J Biol Chem 278(23): 20946-20953.

Tretter, T., Pereira, F.P., Ulucan, O., Helms, V., Allan, S., Kalies, K.U., Römisch, K. (2013) ERAD and protein import defects in a sec61 mutant lacking ER-lumenal loop 7. BMC Cell Biol 14: 56.

Van den Berg, B., Clemons, W.M., Jr., Collinson, I., Modis, Y., Hartmann, E., Harrison, S.C., Rapoport, T.A. (2004) X-ray structure of a protein-conducting channel. Nature 427(6969): 36-44.

Varki, A., Cummings, R.D., Esko, J.D., Freeze, H.H., Stanley, P., Bertozzi, C.R., Hart, G.W., Etzler, M.E. (2009) Essentials of Glycobiology, 2nd edition. Cold Spring Harbor Laboratory Press. 784 p.

Vembar, S.S., Brodsky, J.L. (2008) One step at a time: endoplasmic reticulum-associated degradation. Nat Rev Mol Cell Biol 9(12): 944-957.

Vitale, A., Denecke, J. (1999) The ER - Gateway of the secretory pathway. The Plant Cell 11(4): 615-628.

Walter, P., Ron, D. (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334(6059): 1081-1086.

Ware, F.E., Vassilakos, A., Peterson, P.A., Jackson, M.R., Lehrman, M.A., Williams, D.B. (1995) The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J Biol Chem 270(9): 4697-4704.

Wiertz, E.J., Tortorella, D., Bogyo, M., Yu, J., Mothes, W., Jones, T.R., Rapoport, T.A., Ploegh, H.L. (1996) Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature 384(6608): 432-438.

Wilkinson, B.M., Critchley, A.J., Stirling, C.J. (1996) Determination of the transmembrane topology of yeast Sec61p, an essential component of the endoplasmic reticulum translocation complex. J Biol Chem 271(41): 25590-25597.

Yan, A., Lennarz, W.J. (2005) Two oligosaccharyl transferase complexes exist in yeast and associate with two different translocons. Glycobiology 15(12): 1407-1415.

Zhao, X., Jäntti, Z. (2009) Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit. BMC Cell Biol 10: 76

Zimmermann, R., Eyrisch, S., Ahmad, M., Helms, V. (2011) Protein translocation across the ER membrane. Biochim Biophys Acta 1808(3): 912-924.

Title Image: https://microbewiki.kenyon.edu/index.php/Saccharomyces_cerevisiae_NEU/2011

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VII. Abbreviations

3gpαf N-glycosylated α-factor precursor a.a Amino acid a.u. Arbitrary Unit Ala Alanine Amp Ampicillin Asn Asparagine APS Ammonium Persulfate ATP Adenosine Triphosphate bp Base Pair CAA Casamino Acids Cdc48p Cell Division Cycle ConA Concanavalin A COPI/II Coat Protein Complex I/II CPY Carboxypeptidase Y CPY* Mutant Carboxypeptidase Y (ERAD substrate in yeast) C-terminus Carboxy-terminus Cys Cysteine Da Dalton Der1p Degradation in the ER DMSO Dimethylsulfoxide DNA Deoxyribonucleic Acid Dol-P-P Dolichol diphosphate DTT Dithiothreitol ECL Enhanced Chemiluminescence E. coli Escherichia coli EDEM ER degradation-enhancing 1,2-mannosidase-like protein EDTA Ethylenediamine Tetraacetic Acid e.g. for example ER Endoplasmic Reticulum ERAD ER-Associated Degradation ERAD-C ERAD for cytosolic misfolded domains ERAD-L ERAD for lumenal misfolded domains ERAD-M ERAD for intramembrane misfolded domains ERGIC ER-Golgi Intermediate compartment ERQC ER Quality Control EtOH Ethanol Get3 Guided Entry of Tail-anchored Proteins 3 Glc Glucose GlcNAc N-acetylglucosamine Gls1/2p Glucosidase I/II GPI Glycosylphosphatidylinositol GRP94 Glucose-regulated protein 94 GT Glucosyltransferase GTP Guanosine Triphosphate

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h Hour(s) H2O Water HC Heavy Chain HCl Hydrochloric Acid HMGR 3-hydroxy-3-methylglutaryl acetyl-coenzyme-A Hrd1 HMG-coA Reductase Degradation HRP Horseradish Peroxidase Hyl Hydroxylysine IgG Immunoglobulin G IP Immunoprecipitation kDa Kilodalton kPa Kilopascal LB Lysogeny Broth Leu Leucine LiAc Lithium acetate Man Mannose mCPY mature form of Carboxypeptidase Y Met Methionine MetOH Methanol MES 2-(N-morpholino)ethanesulfonic Acid MgCl2 Magnesium Chloride min Minute(s) MOPS 3-(N-morpholino)propanesulfonic Acid mRNA Messenger RNA NC Nascent Chain NaCl Sodium Chloride NaH2PO4 Sodium Dihydrogen Phosphate Na2HPO4 Disodium Hydrogen Phosphate NaN3 Sodium Azide N-Terminus Amino-terminus OD Optical Density o.N. over night OST Oligosaccharyl-Transferase PAGE Polyacrylamide Gel Electrophoresis pCPY pre-Carboxypeptidase Y p1CPY ER form of Carboxypeptidase Y p2CPY Golgi form of Carboxypeptidase Y PDI Protein Disulfide Isomerase pDNA plasmid DNA PEG Polyethylene glycol PMSF Phenylmethanesulfonyl fluoride ppCPY prepro-Carboxypeptidase Pro Proline Ref. Refer to RNA Ribonucleic acid RNase Ribonuclease RP Regulatory particle, 19S RP of the proteasome rpm Rounds per minute

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Rpn12p Proteasome Regulatory Particle Non-ATPase RT Room Temperature Sbh1p Sec61 beta homologue 1 (also SEB) S. cerevisiae Saccharomyces cerevisiae SDS Sodium Dodecyl Sulfate SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis Sec61p Secretory Ser Serine SH- Sulfhydryl SNARE Soluble N-ethylmaleimide-sensitive-factor Attachment Protein Receptor SP Signal Peptidase SQLE squalene monooxygenase (Erg1 in yeast) SR Signal Recognition Particle Receptor SRP Signal Recognition Particle Ssh1p Sec61 Homologue Sss1p Sec61 Suppressor TA Tail-anchored TBS Tris-Buffered Saline TBS-T TBS + 1% Tween TE Tris-EDTA TEMED N,N,N’,N’-Tetramethylethan-1.2-Diamine TGN Trans-Golgi Network Thr Threonine TM Transmembrane TMD Transmembrane Domain TmDM Transmembrane Double Mutant TRAMp Translocation-Associated Membrane Protein Tris-HCl Tris(hydroxymethyl)aminomethane Hydrochloride Tris Tris(hydroxymethyl)aminomethane TX-100 Triton X-100 UPR Unfolded Protein Response Ura Uracil WB Western Blot Wbp1p Wheat Germ Agglutinin-Binding Protein 1 w/o Without WT Wild-Type YNB Yeast Nitrogen Base YP Yeast Peptone YPD Yeast Peptone Dextrose

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VIII. Annex

1. AUXOTROPHIC AMINO-ACIDS

Table 15 - Composition of Synthetic Complete amino acid drop-out mixture* for S. cerevisiae

Formula Quantity (mg/L)

Adenine 18

L-Alanine 76

L-Arginine HCl 76

L-Asparagine 76

Aspartic Acid 76

L-Cysteine 76

L-Glutamine 76

L-Glutamic Acid 76

Glycine 76

L-Histidine 76

myo-Inositol 76

L-Isoleucine 76

L-Leucine 380

L-Lysine 76

L-Methionine 76

para-Aminobenzoic Acid 8

L-Phenylalanine 76

L-Proline 76

L-Serine 76

L-Threonine 76

L-Tryptophan 76

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L-Tyrosine 76

Uracil 76

L-Valine 76

2. SUPPLEMENTARY FIGURES

Figure 15 - Degradation of CPY* in SBH1 mutant strains over time. (A) Cells were grown at 30°C to an OD600=2 in YPD medium. Cycloheximide was added to inhibit translation, and samples were taken every 15 min for 60 min. Whole-cell extracts were prepared and proteins were resolved on SDS-PAGE. They were transferred onto nitrocellulose membranes and immunoblotted with CPY and Rpn12 as a loading control. (B) Quantification was performed using ImageJ.

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Figure 16 - Import of CPY in SBH1 mutant strains. Transformants and the corresponding WT were grown at 30°C to an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-PAGE. After transfer on nitrocellulose membrane, they were immunoblotted with anti-CPY antibody.

Figure 17 - Import of CPY in SBH1 combined mutant strains. Transformants and the corresponding WT were grown at 30°C to an OD600=2, cell-extracts were prepared from those strains and proteins resolved on SDS-PAGE. After transfer on nitrocellulose membrane, they were immunoblotted with anti-CPY antibody.

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IX. Acknowledgements

First of all, I would like to thank Prof. Dr. Karin Römisch. By accepting my Bachelorthesis in her lab, and proposing this interesting subject, she made me discover a lot of new informations on this part of microbiology and techniques. I also would like to thank her for her help, her advices and her support.

I would also like to thank Joseph Schacherer from the University of Strasbourg for being the examiner of my thesis.

This Bachelorthesis was only possible, because I came to Saarbrücken to achieve a French-German Bachelor, so I also want to thank Joern Pütz, Manfred Schmitt and Björn Diehl for making this happen and for allowing me to live such an amazing adventure. Naturally, I also thank the University of Strasbourg, the University of Saarland, the French-German University (UFA/DFH) and the IdEx for their financial support.

I warmly thank the wonderful lab team, and particularly my two wonderful supervisors, Francesco Elia and Cristina Lupusella. Francesco, you were always there to help me, explain me, and make me do mental arithmetic, and I’d like to say “grazie” for that (except the arithmetic), and Cristina, thank you for all your help and your patience, and for being there to allow me to speak German instead of English.

I also would like to thank Julien Simon, for generating the mutants that I used in a second part of my thesis, and for all the funny moments at lunch when he couldn’t stop laughing and we were forced to laugh too.

Maybe I could thank Fábio Pereira too, for reading my thesis, even though he wasn’t my supervisor, for his help too, and for making me feel less crazy when I talked to him !

My family supported me during these three years of Bachelor, morally as well as financially, so I wanted to thank them for that, and particularly for having been able to boost me when I needed it.

I would like to particularly thank my brother, Yolas, for giving me the force to go on, and for teaching me this year that « it always seems impossible until it’s done ». Well, it’s done now, and it wasn’t impossible after all.

I am also grateful to my grandparents on my father’s side for their financial support during all my Bachelor and for their presence (e.g. through postcards) despite the distance. I’m also grateful to the rest of my family for their support.

I finally wanted to acknowledge two of my friends that were always there when I needed them, and that supported me, advised me, whom I really had fun with : Mathilde and Caroline.

Thank you very much ! - Danke sehr ! - Grazie mille !

Muito obrigada ! - ¡ Muchas gracias ! - Merci beaucoup !

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I hereby confirm that I have written this thesis on my own and that I have not used any other media or materials than the ones referred to in this thesis. I confirm that the submitted electronic version is identical to this printed version.

Saarbrücken, _____________________________

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Bachelor-thesis Antinéa BABARIT