Functional studies of nuclear envelope-associated proteins in

Saccharomyces cerevisiae

Ida Olsson

Stockholm University

© Ida Olsson, Stockholm 2008 ISBN 978-91-7155-666-0, pp 1-58 Typesetting: Intellecta Docusys Printed in Sweden by Universitetsservice US-AB, Stockholm 2008 Distributor: Department of Biochemistry and Biophysics, Stockholm University

To Carl with love

ABSTRACT

Proteins of the nuclear envelope play important roles in a variety of cellular processes e.g. transport of proteins between the nucleus and cytoplasm, co-ordination of nuclear and cytoplasmic events, anchoring of chromatin to the nuclear periphery and regulation of transcription. Defects in proteins of the nuclear envelope and the nuclear pore complexes have been related to a number of human diseases. To understand the cellular functions in which nuclear envelope proteins participate it is crucial to map the functions of these proteins. The present study was done in order to characterize the role of three different proteins in functions related to the nuclear envelope in the yeast Saccharo-myces cerevisiae. The arginine methyltransferase Rmt2 was demonstrated to associate with proteins of the nuclear pore complexes and to influence nu-clear export. In addition, Rmt2 was found to interact with the Lsm4 protein involved in RNA degradation, splicing and ribosome biosynthesis. These results provide support for a role of Rmt2 at the nuclear periphery and poten-tially in nuclear transport and RNA processing. The integral membrane pro-tein Cwh43 was localized to the inner nuclear membrane and was also found at the nucleolus. A nuclear function for Cwh43 was demonstrated by its abil-ity to bind DNA in vitro. A link to nucleolar functions was demonstrated by genetic analysis. Furthermore, Cwh43 is interacting with signalling path-ways perhaps acting as a sensor for signals transmitted from the cytoplasm to the nucleus. The Myr1 protein was found to be membrane-associated and to interact with proteins involved in vesicular traffic. Overexpression of Myr1 affects nuclear morphology and nuclear pore distribution suggesting a function in membrane dynamics. In conclusion, the presented results aid in a deeper understanding of func-tions related to the nuclear envelope in revealing a novel link between argin-ine methylation and the nuclear periphery, identifying a novel inner nuclear membrane protein and a new membrane-associated protein.

LIST OF PUBLICATIONS

The thesis is based on the following publications, which will be referred to by their Roman numerals.

I Olsson I., Berrez J.-M,. Leipus A., Östlund C. and Mutvei

A. (2007) The arginine methyltransferase Rmt2 is enriched in the nucleus and co-purifies with the nuclear porins Nup49, Nup57 and Nup100. Exp. Cell Res. 313:1778-89

II Olsson I. and Mutvei A. (2008) The arginine methyltrans-

ferase Rmt2 specifically associates with FG-nucleoporins – implications for a function in nuclear transport. Manuscript.

III Leipus A., Olsson I., Berrez J. -M., Hultenby K., Östlund

C. and Mutvei A. (2008) Cwh43 is an evolutionary con-served polytopic inner nuclear membrane protein. Submitted to J. Cell Biol..

IV Georgiev A., Leipus A., Olsson I., Berrez J.-M. and Mutvei

A. (2008) Characterization of MYR1, a dosage suppressor of YPT6 and RIC1 deficient mutants. Curr. Genet. 53:235-247

Papers I and IV were reprinted with permissions from the publishers.

Additional publications Lebens M., Sun J. B., Sadeghi H., Bäckström M., Olsson I., Mielcarek N., Li B. L., Capron A., Czerkinsky C. and Holmgren J. (2003) A mucosally administered recombinant fusion protein vaccine against schistosomiasis protecting against immunopathology and infection. Vaccine 21 (5-6) 514-20

TABLE OF CONTENTS

1. INTRODUCTION ....................................................................................11 1.1 General introduction...........................................................................11 1.2 Yeast as a model organism.................................................................12 1.3 Functional organization of the nucleus ..............................................13

1.3.1 The nuclear envelope..................................................................13 1.3.2 The nuclear envelope proteome..................................................15 1.3.3 The Nuclear Pore Complex ........................................................17 1.3.4 Nucleocytoplasmic transport ......................................................19 1.3.5 Targeting of proteins to the nuclear envelope ............................20 1.3.6 Chromatin organization and DNA repair....................................21 1.3.7 Function of the nuclear envelope in chromatin organization and gene regulation ....................................................................................22 1.3.8 Role of the nuclear envelope in diseases ....................................22 1.3.9 The nucleolus..............................................................................23

1.4 PKC signalling ...................................................................................24 1.4.1 Cwh43.........................................................................................24

1.5 Post-translational modifications .........................................................25 1.5.1 Arginine methylation ..................................................................25 1.5.2 Cellular functions of arginine methylation .................................28 1.5.2 Rmt2 ...........................................................................................30

1.6 Vesicular trafficking...........................................................................31 1.6.1 Rab/Ypt proteins.........................................................................32 1.6.2 Ypt6 ............................................................................................32

2. THE PRESENT STUDY ..........................................................................34 2.1 Aims of the study ...............................................................................34 2.2 Results and discussion........................................................................35

2.2.1 Screen for nuclear membrane associated proteins ......................35 2.2.2 The arginine methyltransferase Rmt2 interacts with nucleo-porins (papers I and II) ...................................................................................35 2.2.3 Cwh43 is an integral membrane protein of the inner nuclear membrane (paper III) ...........................................................................38 2.2.4 Myr1is a novel membrane associated protein (paper IV) ...........40

3. CONCLUDING REMARKS....................................................................43

4. FUTURE PERSPECTIVES......................................................................44

5. SAMMANFATTNING.............................................................................46

6. ACKNOWLEDGEMENTS......................................................................47

7. REFERENCES .........................................................................................49

LIST OF ABBREVIATIONS

AdoMet S-adenosyl-L-methionine BMP bone morphogenic protein CWH43 calcoflour white-hypersensitive protein ER endoplasmic reticulum EM electron microscopy GAL galactose GAMT guanidinoacetate methyltransferase GAP GTPase activating protein GEF guanine nucleotide exchange factor GFP green fluoresent protein GTP guanosine 5´- triphosphate INM inner nuclear membrane Kap karyopherin LAP lamina-associated peptide LBR lamin B-receptor MAP mitogen-activated protein MEGA10 decanoyl-N-methylglucamide MYR1 multicopy suppressor of ypt6Δ and ric1Δ NE nuclear envelope NES nuclear export signal NET nuclear envelope transmembrane protein NLS nuclear import signal NPC nuclear pore complex NR nucleoplasmic reticulum ONM outer nuclear membrane ORF open reading frame PKC protein kinase C PM plasma membrane PNS perinuclear space PRMT protein arginine methyltransferase RMT2 protein arginine methyltransferase Smad small mothers against decapentaplegic SNARE soluble N-ethylmaleimide-sensitive factor

attachment protein receptor SPB spindle pole body TAP tandem affinity purification

TGFβ transforming growth factor-β YPT6 yeast protein two

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1. INTRODUCTION

1.1 General introduction Proteins, from the Greek word πρώτα ("prota") meaning "of primary impor-tance", are essential components of living cells. They are large organic com-pounds build by long stretches of amino acids joined by polypeptide bonds. The information specifying the sequence of the amino acids is encoded by a gene. Proteins function as catalysts of chemical reactions, provide structural support to cells and organs, and are involved in immune response, transport and a variety of other cellular processes. To understand how cells work and communicate it is of fundamental importance to understand the function of proteins. To achieve this, biochemical and molecular biological tools are used to evaluate the chemical properties, structures, cellular localizations and binding partners of proteins. Genetic tools are used to analyse the cellular pathways in which the proteins work. An important feature of eukaryotic cells is the presence of membrane-bound compartments. The major compartment is the nucleus that encloses the ge-nome. This thesis describes the functions of three proteins associated with the nucleus and the cellular membranes in the budding yeast Saccharomyces cerevisiae. To elucidate the functions of these proteins comprehensive analyses of their localizations, biochemical properties and binding partners have been undertaken. From those studies these proteins can be linked to RNA processing, nuclear transport, signalling, DNA metabolism, protein translation and membrane biogenesis.

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1.2 Yeast as a model organism Yeasts are unicellular fungi widespread in nature. Around 80 000 fungal species are known today, although the expected number of fungi is thought to be in the range of millions (1). Yeasts live on plant tissues, in soil and water and are also found in parasitic relationships with animals. Yeasts have major economic, social and health significance in human culture. They have been used for production of alcoholic beverages and dough leavening since 2000-6000 BC and in modern times for production of biopharmaceutical agents. Yeasts are in general non-pathogenic although some yeast species such as Candida albicans may cause infections, especially in immunocom-promised individuals (2). In molecular biology studies the budding yeast Saccharomyces cerevisiae, used in brewing and baking, and the fission yeast Schizosaccharomyces pombe are widely utilized as eukaryotic model systems. Yeasts have signifi-cantly contributed to the biological knowledge of today and are important for functional genomics, proteomics and systems biology. Yeasts are also used as model systems for human diseases, e.g. neurodegenerative diseases (3). There are several advantages in using yeasts as model organisms. Yeasts are easy to grow and manipulate genetically. Yeasts have a typical eukaryotic cell organization (Figure 1) and share many biochemical and genetic features with higher eukaryotes (4). The budding yeast differs slightly from higher eukaryotes due to a unique mode of reproduction, an asymmetrical cell divi-sion, a cell wall and a closed mitosis in which the nuclear membranes remain intact around the nucleus during cell division (2). S. cerevisiae was the first eukaryote to be sequenced (5) and contains about 6000 genes on 16 chromosomes. Of those around 4000 genes have been functionally characterized (6). Over 30% of the yeast genes have homo-logues among human genes of known function (7). S. cerevisiae grows by budding and is generally kept as a haploid in the laboratory. It also has a diploid sexual cycle where haploid cells of opposite mating types (a or α) can mate and form diploids (a/α). The diploids can be induced to enter meio-sis and sporulate. S. cerevisiae naturally harbours a circular plasmid, called 2 μ or high copy plasmid, which appears in about 60 copies in the nucleoplasm and is useful for construction of cloning vectors (2).

Figure 1. Overview of the yeast cell organization. NE nuclear envelope; PM plasma membrane; Mito mitochondrion; ER endoplasmic reticulum.

1.3 Functional organization of the nucleus The nucleus is the core organelle of eukaryotic cells. It harbours the genome and is the site of DNA replication and transcription, mRNA processing, rRNA synthesis and ribosome assembly. The nucleus has a complex and highly organized architecture. It is surrounded by the nuclear envelope, con-sisting of a double membrane, which separates the nucleus from the cyto-plasm. The nuclear envelope is acting as a selective barrier to regulate the traffic into and out from the nucleus and is providing a structural framework for the nucleus. In the nucleoplasm, the genome is arranged into chromo-some territories and chromatin domains. The nucleus also contains special-ized compartments, called nuclear bodies, such as the nucleolus where rRNA is processed. The nucleus is a dynamic organelle and there is a rapid ex-change of proteins and other macromolecules both between the nucleus and the cytoplasm and between the nuclear compartments and the nucleoplasm. In addition, mammalian nuclei undergo cycles of disassembling and reas-sembling during the mitosis (reviewed in (8,9)).

1.3.1 The nuclear envelope The nuclear envelope (NE) consists of the outer nuclear membrane (ONM), which faces the cytoplasm and is continuous with the ER membrane, and the inner nuclear membrane (INM), which faces the nucleoplasm. The ONM and INM are separated by the perinuclear space (PNS). The INM and ONM are connected at the pore membrane domains where they form channels that

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are occupied by the nuclear pore complexes (NPCs) regulating the traffic between the nucleus and the cytoplasm. The inner and outer nuclear mem-branes have been suggested to have essentially the same composition of lipids, but have distinct protein composition. The INM has a unique set of integral membrane proteins, while the ONM shares properties with the ER and is studded with ribosomes. The PNS is an extension of the ER lumen (reviewed in (10, 11) (Figure. 2). In metazoan (multicellular species), a filamentous meshwork, the nuclear lamina, which consists of a thick protein network of lamin fibres and lamin-associated proteins, underlines the INM and individual lamins are also found in foci in the nucleoplasm. The lamina system provides mechanical stabiliza-tion to cells, serves as anchor for chromatin and form complexes with pro-teins in the INM. Also, the lamina is linked to the cytoplasmic cytoskeleton (reviewed in (12)). No lamin homologues have been detected in plants or yeast, although lamin paralogues have been suggested to exist in plants (13, 14). It is believed that the lack of a lamina system in unicellular species is due to the closed mitosis, the small nuclei and genomes and the presence of cell walls (reviewed in (15, 16)). In yeast, the NE protein Esc1 tethers chromatin to the nuclear pe-riphery and is thought to play a role analogous to the lamina-associated pro-teins that attach the chromatin to the nuclear periphery (17, 18). Although yeast does not have the lamina system the mechanisms that regulate the nu-clear shape are likely similar in yeast and mammalian cells (19). Interest-ingly, nuclear lamins expressed in yeast localize to the nucleus (20). In addi-tion, the Esc1 protein seems to modify the nuclear structure in a similar manner as lamina in higher eukaryotes (21). In yeast, changes in the morphology of the nuclear membranes can be ob-served in response to pheromones (22) and metabolic changes (23). In higher eukaryotes nuclear membranes can form a dynamic membranous network called the nucleoplasmic reticulum, NR (24), consisting of channels and invaginations containing integral membrane proteins and luminal proteins from the ER, nuclear pore complexes, lamins and cytosolic proteins (25). Some of these channels are close to the nucleoli. The presence of the NR is suggested to be a way to increase transport and communication between the cytoplasm and the nucleus (26). Formation of the NR is regulated by the CTP: phosphocholine cytidylyltransferase (CCT) α, involved in phospho-lipid synthesis, and the lamina A and B (27).

Figure 2. Overview of the nuclear envelope (NE) in metazoan cells. Shown are the inner nuclear membrane (INM) and outer nuclear membrane (ONM) separated by the perinuclear space (PNS). The ONM is continuous with the endoplasmic re-ticulum (ER). The lamina is underlining the INM. LBR, LAP2, LAP1, Emerin, SUN1/2 and MAN1 are examples of integral membrane proteins of the NE. Via Nesprin1/2 and SUN1/2, the lamina is connected to the cytoskeleton. HP1 and BAF are examples of proteins associated with both the chromatin and NE proteins. The NE is penetrated by the nuclear pore complexes (NPC) regulating transport of mac-romolecules into and out from the nucleus. Reprinted from (10) with permission from AAAS.

1.3.2 The nuclear envelope proteome The nuclear envelope has long been thought to be a passive membrane bar-rier between the nucleus and the cytoplasm. Apart from the nuclear pore complexes, rather little is known about the protein composition of the nu-clear envelope. Recent studies have revealed that the NE plays an active role in nuclear events and that NE proteins participate in several important proc-esses such as maintenance of the nuclear structure, anchoring of the chroma-tin to the nuclear periphery, transcriptional regulation, cell cycle events and signalling (reviewed in (10, 28)). Knowledge about the protein composition of the nuclear envelope has started to emerge from proteomic screens and homology searches (29-31). In one screen, 148 NE proteins were found (30) and in another 566 proteins (31). Around 20 mammalian transmembrane NE proteins have been characterized, among them the LBR, LAP1, LAP2,

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Emerin, MAN1, Nurim, LUMA, Nesprin 1, Nesprin 2, Sun 1 and Sun 2 pro-teins (Table 1). The precise functions of these proteins have not been com-pletely revealed. Several of the characterized NE proteins are components of different multi-protein complexes and some of them have been assigned enzymatic activities. The LBR (lamin B-receptor) was identified by its bind-ing to lamin B1 (32) and has also been found to interact with DNA and the heterochromatin protein HP-1 (reviewed in (33)). LBR is homologues to the yeast sterol C-14 reductase, Erg24, and exhibits enzymatic activity. The LAP2β (lamina-associated peptide) has functions in nuclear growth (34) and transcription (35). The Nesprin protein family together with the SUN proteins form a translumenal complex that links the lamina with the cy-toskeleton (36). The MAN1 protein interacts with Smad-proteins in the TGFβ-Smad signalling pathway leading to transcriptional repression of TGFβ−, activin- and BMP- responsive genes (37). In addition, receptors for calcium uptake have been identified in the inner and outer nuclear mem-branes (38).

Table 1. Some of the characterised integral nuclear membrane proteins in mammalian and yeast (S. cerevisiae) cells. NE proteins in mammalian cells

Reference

NE proteins in S. cerevisiae

Reference

Emerin (39) Apq1 (40) gp210 (41) Asi1 (42) LBR (32) Asi2 (43) LAP1 (44) Asi3 (43) LAP2 (45) Brl1 (46) LEM2 (47) Brr6 (48) LUMA (30) Heh1 (49) MAN1 (50) Heh2 (49) Nesprin 1 (51) Kar1 (52) Nesprin 2 (51) Mps2 (53) NET3 (31) Mps3 (54) NET4 (31) Nem1 (55) NET8 (31) Ndc1 (56) NET26 (31) POM34 (57) NET31 (31) POM152 (58) NET39 (31) Snl1 (59) NET51 (31) Spo7 (55) NET56 (31) Nurim (60) POM121 (61) SUN1 (30) SUN2 (62)

UNCL (63)

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Several NE proteins have also been identified and characterized in yeast (Table 1). The Asi1, Asi2 and Asi3 proteins are involved in the regulation of the transcription factors Stp1 and Stp2 (43). The yeast homologue of the mammalian SUN protein, Mps3, has a role in spindle-pole body duplication (54), in telomere clustering in mitotic cells (64) and in anchoring telomeres to the nuclear periphery (65). Two orthologues of the mammalian MAN1 and LEM2 proteins have been identified in yeast, Heh1/Src1 and Heh2 (49). Brr6 and Brl1 are two proteins associated with the ER and NE membranes involved in nuclear transport (48, 46). Apq12 is another integral membrane protein of the ER and NE, which affects membrane dynamics (40). In yeast, the spindle pole body (SPB) is localized and tethered to the NE. This is me-diated by the membrane proteins Ndc1, Mps2, Mps3 and Kar1 (reviewed in (66)). The Cut8 protein in fission yeast is involved in proteasome tethering and DNA double strand-break repair (67, 68).

1.3.3 The Nuclear Pore Complex The spatial separation of the nucleus from the cytoplasm requires exchange of proteins, RNAs and other molecules across the NE. This is achieved through large supramolecular assemblies called nuclear pore complexes (NPC) embedded in the nuclear membranes. The NPCs support both passive diffusion of small molecules (<40 kDa in size) and receptor-mediated trans-port of proteins and RNAs (>40 kDa in size). Each NPC consists of multiple copies of ~ 30 distinct proteins (nucleoporins or Nups) of which several are conserved from yeast to mammals. In yeast, the mass of the NPC is around 44-66 MDa and in vertebrates 60-125 MDa (57, 69). The general structure of the NPC is conserved between species, although differences occur in size and individual nucleoporins. The composition of NPCs may also differ be-tween tissues and cell types and during the cell cycle (reviewed in (70)). The distribution of NPCs is not equal around the nucleus, they are clustered into certain regions (71). By transmission and scanning EM and cryo-electron tomography, the structure of the NPC has been determined to consist of a cylinder shaped central framework (also called the spoke complex), eight cytoplasmic filaments and a nuclear basket composed of eight filaments that form a ring structure. The central pore channel is enclosed by the peripheral channels and function in the traffic of molecules between the nucleus and the cytoplasm (reviewed in (72))(Figure 3).

The nucleoporins can be divided into three classes. The transmembrane nu-cleoporins are considered to anchor the NPCs to the pore membrane in the nuclear envelope. A second class of nucleoporins, called structural nucleo-porins, is expected to directly connect to the transmembrane nucleoporins and to be important for the architecture of the NPC. These proteins give the NPC shape and strength and provide a scaffold for the third class of proteins, the peripheral nucleoporins. This third class of nucleoporins is collectively

named FG-nucleoporins because they contain hydrophobic repeats of a phenylalanine-glycine (FG) motif, or the variant motives FXFG and GLFG, disrupted by hydrophilic linker sequences. The FG-nucleoporins are in-volved in the transport of molecules through the NPCs by interacting with different transport receptors. The FG-domains are natively unfolded and have been suggested to be mobile within the NPCs (reviewed in (70, 72)).

Figure 3. A three-dimensional representation of the consensus model of the nuclear pore complex (NPC). The structural components of the NPC consist of the central framework, the cytoplasmic ring moiety and the cytoplasmic fibrils extend-ing into the cytoplasm, and the nuclear ring moiety and the nuclear basket. Adapted from (73) by permission from Macmillan Publishers Ltd.

How the translocation through the NPC works and how the FG-domains contribute to the selective gating of the NPC is not clear. Several models have been proposed based on views of the permeability barrier as either physical or energetic or both (reviewed in (70, 74)). There are studies show-ing that different transport receptors have different binding affinities for certain nucleoporins (75). Favoured association of the receptors with particu-lar nucleoporins may thus regulate the transport. Recent studies show that deletion of specific combinations of FG-domains had various impacts on different transport receptors suggesting the existence of several independent transport pathways through the NPCs (76, 77).

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1.3.4 Nucleocytoplasmic transport The transport of molecules through the NPCs is mediated by transport recep-tors, which recognize their cargoes in the cytoplasm or the nucleus and carry them through the NPC by interactions with the nucleoporins. The largest group of transport receptors is the conserved karyopherin-β/importin-β (Kapβ/Impβ)-family. These receptors bind to nuclear localization signals (NLS) or nuclear export sequences (NES) directly in a cargo or through an adapter protein. The Kaps can also bind to nucleotide sequences in RNA cargoes. Kaps that import cargoes into the nucleus are referred to as im-portins and Kaps that mediate export from the nucleus are called exportins. In human cells there are 20 known members of the Kapβ family and in yeast there are 14 members. The different transport receptors appear to bind to certain set of substrates (reviewed in (78)).

The directionality of the transport is regulated by the small GTPase Ran (Gsp1 in S. cerevisiae) that exists in a GTP-bound state in the nucleus and a GDP-bound state in the cytoplasm. Two Ran regulators, RCC/RanGEF (Ran-GDP-exchange factor; Prp20 in S. cerevisiae) in the nucleus and Ran-GAP1 (Ran-GTPase activating protein; Rna1 in S. cerevisiae) in the cyto-plasm create the RanGTP-RanGDP gradient across the nuclear membranes. The import receptors, importins, pick up their cargoes in the cytoplasm, translocate them through the NPC and release them in the nucleus upon binding to RanGTP. The export receptors, exportins, carry their cargoes together with RanGTP and release them in the cytoplasm upon hydrolysis of RanGTP to RanGDP by the RanGAP protein (reviewed in (78, 79)). There are also transport receptors besides the Kapβ family. Ntf2 is a trans-port receptor for the import of RanGDP and Mex67 (Nfx1/Tap in metazoan) is the transport receptor for the mRNA export from the nucleus. The Mex67 receptor works independently of the Ran gradient. The export of the 60S ribosomal subunit in yeast requires several transport receptors and there are also examples of receptor-independent transport (reviewed in (74)). The NLS and NES signals that are recognized by the Kapβ family of trans-port receptors have been identified and characterized in only a few cases and few consensus sequences have been found. The classical NLS consists of a short basic peptide and was originally identified in the SV40 large tumour antigen (80). The bipartite classical NLS consists of two stretches of basic residues separated by about 10 linker residues (81). The NES signal contains a leucine-rich stretch of amino acid residues (reviewed in (82)). Nuclear import mediated by non-classical NLS sequences have been found in the mRNA-binding protein hnRNP A1 (83), in histones (84) and in the Asr1 protein that accumulates in the nucleus upon alcohol-induced stress (85).

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Regulation of the nucleocytoplasmic trafficking is mediated at multiple lev-els comprising the NPC transport channel, the transport receptors and the transport cargos. The control events include regulation of the interactions of transport receptors with the NLS and NES signals on the cargos, posttransla-tional modification of cargos, posttranscriptional regulation of RNAs, regu-lation of transport receptor expression levels, competition among transport receptors for nucleoporin binding sites and regulation of the pore permeabil-ity (reviewed in (74)).

1.3.5 Targeting of proteins to the nuclear envelope Integral membrane proteins are co-translationally integrated into the ER membrane at sites called translocons (86). The proteins are then sorted and delivered by vesicular trafficking to their functional sites at the Golgi com-partments, plasma membrane and other organelles (87). How proteins des-tined for the inner nuclear membrane (INM) actually reach their destination is not well established. One model, the so called diffusion-retention model, proposes that proteins destined for the INM diffuse through the ER mem-branes, the outer nuclear membrane (ONM) and the pore membranes to the INM where they are immobilized by association with DNA or nuclear pro-teins (88). The LBR protein has been propose to reach the INM by this mechanism (89, 90). Recent studies of viral, yeast and mammalian proteins destined for the INM indicate that the sorting of proteins to the INM involve specific signal se-quences, energy and protein-protein interactions (49, 91-94). A novel mem-brane-associated transport receptor, importin-α-16, was recently implicated in the transport of INM proteins from the ER to the INM (95). Additional studies in yeast have implicated a role for the Ran protein and the Kap95 receptor in recruiting proteins to the NPC by a vesicular mechanism for de novo NPC assembly into intact NE (96, 97). Also, mislocalization of nucleo-porins has been observed in mutants of vesicle traffic proteins (98). These observations indicate that vesicular traffic or ER membrane biogenesis could have a role in targeting proteins to the nuclear envelope. Targeting of nuclear membrane proteins after nuclear envelope breakdown during mitosis in higher eukaryotes occurs by different mechanisms than targeting of proteins to the nuclear envelope during biosynthesis of the nu-cleus (reviewed in (99)). During the mitotic phase of the cell cycle, micro-tubules of the mitotic spindle need to reach the chromosomes. In yeast, this is achieved by assembling the spindle inside the nucleus (“closed mitosis”). In metazoan, the spindle is cytoplasmic and the NE breaks down to allow the chromosomes access the spindle. After the chromosome segregation is com-pleted, the NE reassembles again (“open mitosis”). During the breakdown process proteins of the NE reside in the ER, which remains intact. Observa-

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tions have also been made of vesiculation of the NE (reviewed in (100)). The reassembly is an ordered process that includes targeting of membranes to the chromatin, fusion of membranes and eventually insertion of the NPCs. The Ran protein, a member of the Ras-like family of small GTPases regulating the nuclear transport, has an important role in this process (reviewed in (100, 101)).

1.3.6 Chromatin organization and DNA repair The human genome encompasses around 20-35,000 genes and 3.2 billion base pairs (102, The GDB Human Genome Database http://www.gdb.org/). The yeast genome contains 6-7,000 genes and 12.2 million base pairs (Sac-charomyces Genome Database, http://www.yeastgenome.org/). To enclose the whole genome into the nucleus, the DNA is tightly condensed into chro-mosomes, which consist of higher-order chromatin structures. The basic structural unit of the chromatin is made of 146 base pairs of the DNA strand wrapped around a core of histones, forming the nucleosome. Arrays of nu-cleosomes fold into a 30 nm fibre that is further folded into the higher-order structure of the chromatin (103, 104). The folding of chromatin into higher-order structure and the expression of genes are linked to various posttransla-tional modifications, such as acetylation, methylation, phosphorylation and ubiquitination of the histones in the nucleosome core. The chromatin is organized into heterochromatin, a condensed chromatin structure enriched in inactive genes, and euchromatin with a more open con-formation enriched in active genes (reviewed in (102)). Recent studies indi-cate however that although open chromatin has higher gene density this does not need to correlate with high gene expression and vice versa for closed chromatin (105). In the nucleus, the chromosomes are arranged into chromo-some territories, with the high gene density chromosomes in the nuclear interior and the low gene density chromosomes at the nuclear periphery (re-viewed in (102)). In differentiated somatic cells, the vast majority of the chromatin exists as transcriptionally silent heterochromatin while in yeast most of the chromatin is actively transcribed (reviewed in (106)).

The highest priority for living cells is to maintain the integrity of their ge-nomes and thus systems for handling various threats to the DNA have evolved. Damage of the DNA and impaired DNA repair pathways are causa-tive agents in mutagenesis, carcinogenesis and ageing. DNA can be damaged in several ways by environmental agents such as UV light, ionising radiation and pollutants, by-products of the normal cellular metabolism such as reac-tive oxygen species (ROS) and spontaneous lesions of bases such as replica-tion errors or chemical alterations of bases. There are several DNA repair pathways used by cells to overcome DNA lesions including base-excision

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repair (BER), nucleotide excision repair (NER), recombinational repair (HR, EJ) and mismatch repair (reviewed in (107)).

1.3.7 Function of the nuclear envelope in chromatin organization and gene regulation The nuclear envelope plays an important role in the organization of the chromatin, expression of genes and recent studies also suggest a function in genome stability (reviewed in (106)). Silent chromatin is anchored to the nuclear periphery. In yeast this is mediated by the Sir/Esc1 proteins or the yKu proteins, which localize the silent chromatin to foci at the inner nuclear membrane at regions between the NPCs. In metazoan cells, heterochromatin is anchored at the nuclear periphery through the nuclear lamina and associ-ated proteins (106). Active transcription has in several studies been linked to the NPCs and nucleoporins have been coupled to promoter-binding factors and mRNA processing factors (108-110). In addition, the NPCs, and espe-cially the Nup84 complex, have been implicated a role in telomere anchoring and DNA double strand break (DSB) repair (111-113). Several INM proteins interact with transcription factors and other gene-regulatory proteins in-volved in down regulating gene expression, among them the mammalian LAP2β protein that induces deacetylation of histone H4 resulting in gene repression (114, 115).

1.3.8 Role of the nuclear envelope in diseases Mutations in genes encoding nuclear lamins or integral nuclear membrane proteins are associated with a variety of human diseases and disorders. In particular, mutations in the LMNA gene encoding the A-type lamins cause more then 10 different clinical syndromes, collectively named “laminopathies” or “nuclear envelopathies”. These diseases are primarily affecting skeletal and cardiac muscles, adipose tissue, nerves of the periph-eral nervous system or resulting in premature ageing. Also, mutations in genes encoding B-type lamins and the lamin-associated integral membrane proteins Emerin, LBR, MAN1 and Nesprins are linked to diseases. The pre-dicted mechanisms behind these syndromes are that mutations in the lamina and lamina-associated proteins result in fragile and mechanically unstable nuclei, which in turn could result in mechanically fragile cells with contrac-tile dysfunctions. Mutations may also effect the distribution of NPCs, gene regulation, DNA replication and DNA repair (reviewed in (10,116,117)). The morphology of the nucleus is often altered in cancer cells and character-istic changes of the nuclear shape are associated with specific tumours (118). Changes in the shape of the chromatin, nucleoli and other subnuclear struc-tures are also associated with tumours.

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Defects in the regulation of nucleocytoplasmic transport have been found associated with several diseases, in particular cancer. For example, the tran-scription factor NF-κB is mislocalized to the nucleus in many cancer cells such as Hodgkin’s lymphoma and breast tumour cells and cytoplasmic se-questration of the tumour suppressor p53 has been related to breast carcino-mas and neuroblastomas (reviewed in (119)). The observed mislocalizations were due to masking of the NLS and NES signals or mutations in transport receptors. In addition, rearrangements giving rise to fusion proteins between the GLFG nucleoporin NUP98 (orthologues to the yeast nucleoporins Nup100, Nup116 and Nup145N (120)) and the homeobox transcription fac-tor HOX9A have been discovered in acute myelogenous leukaemia (AML). The GLFG repeats have been suggested to promote interactions between NUP98-HOX9A and other transcription factors (119, 121). Mutations of the mammalian nucleoporin ALADIN is related to Triple A syndrome and nu-clear import of proteins involved in DNA repair has been found affected in an ALADIN mutant leading to an accumulation of damaged DNA (122). Overexpression of the transport receptor Kapα has been detected in breast cancer cells (123).

1.3.9 The nucleolus The nucleus is compartmentalized and contains different kinds of so-called nuclear bodies. In contrast to the cytoplasmic compartments such as mito-chondria and Golgi, the nuclear compartments are not surrounded by mem-branes. The major nuclear bodies are the nucleoli, Cajal bodies, gems, splic-ing speckles and promyelocytic leukaemia (PML) bodies (reviewed in (9)). The nucleolus is a subnuclear compartment that forms around the ribosomal RNA (rRNA) genes. The nucleolus can be divided into three morphological regions: the fibrillar centres (FC), the surrounding dense fibrillar compo-nents (DFC) and the granular components (GC). The primary function of the nucleolus is to synthesize rRNA and assemble the ribosomal subunits. The rRNA is post-transcriptionally modified through interactions with small nucleolar ribonucleoproteins (snoRNPs) and proteins and then assembled with ribosomal proteins before export to the cytoplasm. The nucleolus thus contains a large number of proteins related to this processing. In addition, proteins associated with cell cycle control, DNA replication and repair have been found in the nucleolus (reviewed in (124)). In yeast the nucleolus occu-pies around 1/3 of the nuclear volume and divides at the end of anaphase, in contrast to the mammalian nucleolus that is disassembled during mitosis (125). In yeast, the nucleolus is localized close to the inner nuclear mem-brane (126) and opposite to the spindle-pole body (127).

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1.4 PKC signalling All living cells depend on the ability to sense their environment and respond appropriately to changes in their surroundings and thereby survive in a vari-ety of biological niches (reviewed in (128)). Cells use numerous signal-transduction pathways to receive extracellular signals and respond by modi-fying the transcriptional, metabolic and developmental programs. Yeasts are found in a variety of environments and thus have to adapt to a diversity of signals such as pheromones, rapid changes in nutrient availability, light, gases and fluctuations in pH and osmolarity to maximize survival. To withstand osmolarity changes, yeasts are surrounded by rigid cell walls that limit swelling. Responses to cell wall stress are maintained through the cell wall integrity pathway (CWI) that mediates signals from cell surface sensors linked to Rho, a small G protein, which activates a number of effec-tor molecules. This gives rise to signalling cascades that regulate e. g. carbo-hydrate synthesis at the site of the cell wall remodelling, expression of genes involved in cell wall biogenesis, re-organization of the actin cytoskeleton and targeting of secretory vesicles to the growth site (reviewed in (129)). One of the first components identified of the CWI pathway was protein kinase C, PKC, which is one of the Rho effectors. Yeast cells have a single Pkc1 protein, while in mammalian cells there are 10 isoforms of PKC and two PKC-related kinases. The yeast Pkc1 is a functional homologue to the human PKC-eta, an isoform that does not respond to Ca2+ or diacylglycerol as conventional PKCs do. Pkc1 is regulating the Bck1-Mkk1/2-Mpk1 MAP kinase cascade but has also been suggested to have additional substrates (reviewed in (129)). Pkc1 has been found to interact with the enzyme com-plex oligosaccharyltransferse located in the ER, suggesting a function in protein N-glycosylation (130). Elevated rates of mitotic recombination have been observed in mutants lacking Pkc1, suggesting that Pkc1 may regulate DNA metabolism (131). Pkc1 has been genetically related to spindle pole body (SPB) components and proposed a role in SPB duplication (132). In addition, genetic connections have been revealed between Pkc1 and the ATP-dependent chromatin remodelling complex RSC (133).

1.4.1 Cwh43 The CWH43 gene was identified in a screen for mutants sensitive to Cal-coflour white, a chemical causing cell wall perturbations (134). Subsequent studies showed that the cwh43Δ mutant is lethal in combination with a dele-tion of the PKC1 gene, and suggested Cwh43 to function in a pathway paral-lel to the Bck2 branch of the PKC signalling pathway (135). Recent studies have also suggested a role for Cwh43 in remodelling of GPI anchors, since synthesis of ceramide-containing GPI anchors is abolished in cwh43 mutants (136, 137). Analysis of the amino acid sequence of Cwh43 predicts that it

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contains 14-16 transmembrane regions and a hydrophilic C-terminal (135). Cwh43 is a conserved protein, which in higher eukaryotes has evolved into two proteins. The N-terminus of Cwh43 is homologues to the human protein Frag1, while the C-terminus is homologues to the human FLJ21511 (referred to as human Cwh43).

1.5 Post-translational modifications The functions of proteins may be regulated by the introduction of chemical modifications after their synthesis. A variety of posttranslational modifica-tions exist that may change the activity, life span and cellular location of the proteins. Posttranslational modifications may involve removal of a peptide segment, addition of small chemical groups such as phosphoryl-, methyl- and acetyl groups or attachments carbohydrates or lipids (138).

1.5.1 Arginine methylation Methylation of arginine residues was discovered already in 1967 (139) but it is only during the last decade that the functions of this specific methylation have come into the light. Arginine is a positively charged amino acid that mediates hydrogen bonding and amino-aromatic interactions. It can be me-thylated on the internal (δ)- and outer (ω)-nitrogens. The methylation of the outer nitrogens can produce monomethylated or symmetric- or asymmetric dimethylated arginine residues, while methylation of the internal nitrogen only produces monomethylated arginines (reviewed in (140, 141)) (Figure 4). Few studies have addressed the molecular effects of arginine methylation. The addition of methyl groups does not change the charge of arginine but disrupts hydrogen bonding and induces steric hindrance. Arginine methyla-tion could thus modulate intra-or inter-molecular interactions of target pro-teins (reviewed in (142)). It has also been speculated that arginine methyla-tion could disrupt protein folding (143). Arginine methylation is thought to positively or negatively regulate protein-protein, protein-RNA or protein-DNA interactions (144, 145). Arginine methylation is catalysed by a family of protein arginine methyl-transferases (PRMTs) that use S-adenosyl-L-methionine (AdoMet) as the methyl group donor. PRMTs are divided into four groups depending on the nature of the methylated arginine residues they produce. The type I, II and III enzymes all add methyl groups to the outer nitrogens producing mono-methylated arginines. From the monomethylated arginine, the type I en-zymes can also produce asymmetric dimethylated arginines while the type II

produces symmetric dimethylated arginines (Figure 4). Type IV enzymes catalyse formation of monomethylated arginines on the internal nitrogen (reviewed in (141)).

Figure 4. Posttranslational methylation of arginine residues. Arginines can be methylated in four ways catalysed by four different classes of enzymes (Type I-IV). The type I-III enzymes methylate the outer (ω) -nitrogens, while the type IV en-zymes methylate the internal (δ)-nitrogen of arginine residues. PRMT10 and PRMT11 have not been categorized into type I-IV and are omitted from the picture.

In mammalian cells, eleven different arginine methyltransferases PRMT1-11, of type I, II and III, have been identified (reviewed in (146)). In yeast Hmt1, a functional homologue to the mammalian PRMT1 (type I), has been identified (147, 148) together with Hsl7 and Skp1 (149, 150), which are homologues to the mammalian PRMT5 enzyme (type II). The yeast enzyme Rmt2 is the only type IV enzyme identified (151). In fission yeast, a homo-logue of the mammalian PRMT3 enzyme, Rmt3, has been found that has not

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been discovered in budding yeast (152) (Table 2). Arginine methyltrans-ferases have also been found in other species such as flies, fish and plants (153, 154).

Table 2. Protein arginine methyltransferases (PRMTs) identified in mammal-ian and yeast cells and their predicted functions. S.c Saccharomyces cerevisiae, S.p Schizosaccharomyces pombe. Mammalian PRMTs

Type

Functions

Yeast PRMTs

Type

Functions

PRMT1

I

Transcriptional activation, cellular localization, DNA repair, signalling

Hmt1/Rmt1 (S. c)

I mRNA process-ing and export, transcription

PRMT2

I

Transcriptional coactivator, apoptosis, cellular localiza-tion

Rmt3 (S. p)

II

Ribosome assembly

PRMT3

II

Ribosome assembly, RNA metabolism

Hsl7 (S. c)

II

Mitosis

CARM1/PRMT4

I

Transcriptional regulation, muscle differentiation, T cell development, tumori-genesis

Skb1 (S. p)

II

Cell viability and morphol-ogy, mitosis

PRMT5

II

Transcriptional regulation, signal transduction, RNA processing, mitosis, muscle and germ cell differentia-tion, tumorigenesis

Rmt2 (S. c) IV Unknown

PRMT6 I HIV replication, DNA repair

PRMT7 III RNA processing, transcrip-tion

PRMT8 I Unknown PRMT9/FBXO11 II Unknown PRMT10 ? Unknown PRMT11 ? Unknown

Arginine methylation was long thought to be an irreversible modification, but recently the dioxygenase JMJD6 was found to demethylate histones (155). In addition, arginine methylation can be antagonized by deimination. The peptidyl deiminase PAD4 can convert non-methylated and methylated arginine residues in histones to citrulline by deimination (156, 157). How these modifications regulate arginine methylations in different cellular proc-esses has not been determined. The PAD4 has been found to function as a transcriptional repressor (156). Knockout mutants of JMJD6 display devel-opmental defects suggesting an important function in regulating arginine methylation (155).

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PRMTs are present in both the nucleus and the cytoplasm. PRMT1 and PRMT5 localize to the nucleus and the cytoplasm, PRMT6 has predomi-nantly nuclear localization and PRMT8 is localized to the plasma membrane in brain (reviewed in (158)). Disruption of PRMT genes does not have large viability effects on cells in general. However, deletion of PRMT1 causes embryonic lethality (159) and CARM1 knockout mice die at early age (160). Furthermore, deletion of the yeast HMT1 gene is lethal in temperature-sensitive mutants of its substrate Npl3 (148).

1.5.2 Cellular functions of arginine methylation There is an extending list of reported substrates for arginine methyltrans-ferases, which are summarized in Table 3. The substrates are mainly found within RNA-binding proteins, histones and transcription factors. Target sub-strates for PRMTs often contain glycine-and arginine rich sequences such as GAR or RGG motifs. Substrates for CARM1 and PRMT5 have also been found to contain PGM motifs, which are sequences rich in prolines, argini-nes, methionines and glycines (161). There are also a number of substrates without RG-motifs, such as histones (reviewed in (146)). Arginine methylations have been linked to a number of cellular processes such as RNA processing, nucleocytoplasmic trafficking, signal transduction, transcription, DNA repair and chromatin remodelling reviewed (in (140, 142, 146)) (Table 2). For example, the yeast Hmt1 enzyme facilitates nu-cleocytoplasmic shuttling of mRNA-binding proteins (162). In addition, Hmt1 is recruited to transcriptionally active genes and affects transcriptional elongation and recruitment of RNA-processing factors (163). Hmt1 has also been implicated a function in formation of silent chromatin (164). Cytoplas-mic assembly of the core components of the spliceosome, the small nuclear ribonucleotide particles snRNPs, is dependent on arginine methylation (165). Arginine methylation of the DNA damage checkpoint protein MRE11 regu-lates its exonuclease activity suggesting a function of arginine methylation in regulation of DNA damage response (166). The type I enzyme CARM1 is a coactivator for p53-, nuclear receptor - and NF-κΒ-dependent transcription (167, 168) and has a potential role in prostate cancer (169). In addition, PRMT1 and CARM1 have cooperative functions in p53-regulated transcrip-tion (167). Arginine methylation has also been related to a number of human diseases, among them cancer, cardiovascular disease, viral pathogenesis, multiple sclerosis (MS) and spinal muscular atrophy (SMA) (140).

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Table 3. Substrates for arginine methylation. A high number of additional sub-strates have been found and not all PRMTs have been assessed substrates. Some substrates have only been identified as in vitro targets for arginine methyltrans-ferases. S.c. Saccharomyces cerevisiae, S.p. Schizosaccharomyces pombe, mam. Mammalian. References (140, 152, 158, 164, 170, 171). Arg.methyl-transferases Substrates

Chromatin related proteins

RNA associ-ated proteins

DNA repair

Viral protein

Signalling proteins

PRMT1 (mam.)

histone H4 histone H2A RIP140

hnRNP A1, A2 Sam68 Nucleolin RNA helicase A

Mre11

EBNA1

STATs Smad6

PRMT3 (mam.)

S2 Sam68 PABP2

PRMT4/CARM1 (mam.)

histone H3 histone H2A p300 GRIP

PABP1 SmB SAP49

PRMT5 (mam.)

histone H4 histone H3 histone H2A SPT5

SmB SmD1 SmD3

EBNA1 EBNA2

PRMT6 (mam.)

histone H4 histone H3 histone H2A HMGA1a HMGA1b

DNA polβ

HIV-Tat HIV-Rc HIV-Rev

PRMT7 (mam.)

histone H4 histone H3

PRMT9 (mam.)

histone H4 histone H2A

Hmt1 (S. c.)

histone H4

Npl3 Nab2 Hrb1 Hrp1 Gar1 Nop1 Nsr1

Rmt2 (S. c.) L12 Rmt3 (S. c.) S2

1.5.2 Rmt2 The only type IV arginine methyltransferase known today, Rmt2, was origi-nally identified in a study of putative arginine methyltransferases in yeast and was found responsible for the addition of methyl groups to the internal (δ)-nitrogen of arginine residues (172, 151). This specific arginine methyla-tion has yet only been identified in yeast and the amount of the δ-nitrogen methylated arginine derivatives is very low compared to the amount of ω-nitrogen methylated arginines executed by type I-III enzymes (151, 172, 173). Rmt2 thus represents a subgroup of arginine methyltransferases. Rmt2 is conserved among fungi and plants and also shows sequence similarities with the human enzyme guanidinoacetate methyltransferase (GAMT). Se-quence alignment of Rmt2 and its homologues reveals the conserved AdoMet-binding domain in the C-terminal part of the proteins and the pres-ence of ankyrin (ANK) repeats in the N-terminal (Figure 5). The ankyrin repeat is a common protein sequence motif found in a number of different proteins and functions in mediating protein-protein interactions (174).

Figure 5. Schematic representation of the domain organisation of Rmt2 and representative homologues in yeasts, plants and human. N. crassa Neurospora crassa, A. thaliana Arabidopsis thaliana, O. sativa Oryza sativa, GAMT guanidi-noacetate N-methyltransferase.

One substrate has been identified for Rmt2, the ribosomal protein L12 (170), methylated on Arg67. The biological function of the L12 methylation has not yet been established. Large-scale protein-protein interaction studies in yeast have found interactions between Rmt2 and the protein phosphatase Sit4, the nucleoporin Nup116, the meiotic recombination protein Mer2/Rec107, the autophagy protein Atg12 and the uncharacterised ORF YBR261c (175, 176).

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1.6 Vesicular trafficking An important feature of eukaryotic cells is the presence of membrane-bound organelles, such as the endoplasmic reticulum (ER), the Golgi apparatus, endosomes, lysosomes (animal cells) and vacuoles (fungi and plant cells). The traffic of proteins and lipids between the organelles is mediated by transport vesicles, which bud from a donor compartment and fuse with an acceptor compartment. The exocytic pathway is used for transport of newly synthesized proteins from the ER to the Golgi apparatus and then to their final destinations at the extracellular space or at the plasma membrane, vacuoles or lysosomes. The retrograde transport mediates the retrieval of transport components back to the donor compartments. Uptake of nutrients and internalisation of receptors is mediated by the endocytic pathway, which transport their cargoes to the early and late endosomes. The proteins are then either recycled back to the plasma membrane or sorted to the ly-sosomes/vacuoles for degradation (reviewed in (177, 178)) (Figure 6).

Figure 6. Overview of the intracellular transport pathways. Vesicles containing COPII bud of from the ER compartment and fuse with the ER-Golgi intermediate compartment (ERGIC) or the Golgi. From the Golgi compartment vesicles are transported to the PM. Retrograde transport from trans-Golgi network (TGN) to the ER involves COPI. Clathrin coated vesicles with internalised extracellular molecules move from the PM to endosomes and lysosomes. Reprinted from (177) with permis-sion from Elsevier.

The generation of bud vesicles and cargo selection are mediated by protein coats composed of supramolecular assemblies of cytosolic proteins. Various coat complexes exist involved in different steps of vesicle traffic. The target-

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ing and fusion of vesicles with the appropriate compartments are mediate by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein re-ceptors). Vesicle-associated v-SNAREs fuse with target membrane t-SNAREs. There are several families of SNAREs with different distributions and specific combinations of SNAREs are thought to contribute to the speci-ficity of membrane fusions (reviewed in (177, 179)). To achieve vesicle targeting and fusion SNAREs act in collaboration with tethering factors and Rab GTPases (reviewed in (178)).

1.6.1 Rab/Ypt proteins The Rab family of Ras-like GTPases are small monomeric proteins of 20-30 kDa. Eleven members have been identified in S. cerevisiae and around 60 in mammalian cells, depending on tissue and cell type. The Rab proteins cycle between GTP-bound and GDP-bound states, where the GTP-form associates with membranes and the GDP-form is cytosolic. The cycling of GTP-and GDP-bound states is regulated by guanine nucleotide exchange factors (GEFs) that promote binding of GTP and GTPase-activating proteins (GAPs) (reviewed in (178, 180)). A number of proteins are involved in regu-lating membrane attachment and detachment of the Rab proteins. In addi-tion, effector proteins interacting with the GTP-bound state of the Rab pro-teins have been identified that associates with specific Rabs and regulate the different Rab functions. The different Rabs localise to distinct membrane-bound compartments and some of them are tissue specific. The Rabs partici-pate in several steps of the vesicular traffic, in receptor cargo collection dur-ing vesicle formation, in enabling motor proteins to interact with membranes and in the complex events during docking and fusion of transport vesicles with their target membranes (reviewed in (178, 180)). In S. cerevisiae, the Rab protein Ypt1 is involved in mediating ER-Golgi transport; the Ypt3 protein is involved in endocytic recycling and possible intra-Golgi traffic; Ypt5 is involved in Golgi-endosome and PM-endosome traffic; Ypt7 promotes vacuole fusion and Sec24 is involved in delivery of vesicles to the bud (reviewed in (181)).

1.6.2 Ypt6 The yeast Rab GTPase Ypt6 is homologues to the mammalian Rab6 protein and functions in recycling of proteins from endosomes to Golgi, in intra-Golgi retrograde transport and also in Golgi-to-ER transport (182). Ypt6 is activated by the GTPase exchange factor (GEF) Ric1-Rgp1, which is a pe-ripheral membrane protein found in the Golgi. Ypt6 localizes to the Golgi at steady-state, and the Golgi-association is dependent on the presence of Ric1 (183). This suggests the GTP-form of Ypt6 to act on the Golgi. Ypt6 is not an essential protein, but deletion mutants exhibit several phenotypes such as

33

temperature-sensitive growth, defects in secretion of invertase, partial mi-sorting of the vacuolar protease carboxypeptidase Y and mislocalization of the Golgi protein Kex2 to the vacuole (184, 185). Several studies have been done in order to investigate the pathways in which Ypt6 operates. High copy suppressor screens for genes that upon overex-pression can rescue the ts mutant of ypt6Δ at 37ºC have identified the Ypt1 and Imh1/Sys3 proteins (184, 186), the v-SNAREs Gos1 and Ykt6, the t-SNARE activator Sly1, the Ras protein Arl1 (187) and the Golgi membrane protein Sys1 (186).

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2. THE PRESENT STUDY

2.1 Aims of the study The primary aim of this study has been to investigate the functions of pro-teins associated with the nuclear envelope in order to gain a deeper under-standing of the role of the nuclear membranes in the organization of nuclear processes. For this purpose a screen for nuclear envelope associated proteins was conducted in S. cerevisiae and the proteins identified have been ana-lysed with respect to their cellular roles. The specific aims of the separate studies were: Paper I: To investigate the cellular localization and functions of the protein arginine methyltransferase Rmt2. Paper II: To further investigate the function of Rmt2 with respect to its role at the nuclear pore complex. Paper III: To study the function of the integral membrane protein Cwh43. Paper IV: To search for proteins interacting with the Ypt6 pathways and to characterize the Myr1 protein.

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2.2 Results and discussion 2.2.1 Screen for nuclear membrane associated proteins Few proteins associated with the nuclear envelope have been identified and characterized. Using the yeast S. cerevisiae as a model organism, we per-formed a screen for new putative nuclear membrane associated proteins. Yeast nuclei were purified on a sucrose gradient and subjected to treatments with the detergents Triton X-114 (188) or MEGA10. The hydrophobic frac-tions containing membrane-associated proteins were isolated and used for immunization of rabbits. The antibodies produced were subsequently used to screen an expression library in λgt11. Among the proteins identified from the Triton X-114 extract were Rmt2, a type IV arginine methyltransferase, and Cwh43, implicated in PKC signalling pathways. From the screen of the MEGA10 extract Ypt6, a Rab GTPase, was identified.

2.2.2 The arginine methyltransferase Rmt2 interacts with nucleoporins (papers I and II) In this study, the main focus has been to investigate the cellular function of Rmt2 and in particular its role at the nuclear periphery. Although identified as an arginine methyltransferase (151) and demonstrated to be responsible for the arginine methylation of the ribosomal protein L12 (170), the cellular function of Rmt2 has not previously been addressed. To characterize Rmt2, a polyclonal antibody was first raised against recom-binant Rmt2. By immunofluorescence microscopy and subcellular fractiona-tion, Rmt2 was distributed to both the nucleus and the cytoplasm with en-richment in the nucleus. In isolated nuclei, Rmt2 localized to the nuclear periphery in close proximity to and between the NPCs and also to the nu-cleoplasm. Analyses of the biochemical properties of Rmt2 showed that only a small amount of Rmt2 partitioned in the Triton X-114 fraction of isolated nuclei. In further studies the nuclear Rmt2 was not released from membranes by detergent treatment and could only be solubilized by urea treatment. The cytoplasmic Rmt2 on the other hand behaved as a soluble protein. Taken together, this indicates that the nuclear Rmt2 is associated with structural components and not with membranes. However, in the light of the hydro-phobic properties of Rmt2 it cannot be excluded that it may also be associ-ated with membranes. Possibly, Rmt2 associates both with membranes and structural proteins. The various cellular localization and properties displayed by Rmt2 suggest that it has separate functions in different cellular compart-ments. A possibility is that Rmt2 could be sequestered in the cytoplasm and move to the nucleus in order to perform its function (Paper I).

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The previous findings of an interaction between Rmt2 and the nucleoporin Nup116 (175) together with the nuclear localization of Rmt2 made us inter-ested in exploring the association between Rmt2 and NPC. To examine if Rmt2 interacts with other nucleoporins than Nup116, we performed pull-down assays of TAP- (tandem affinity purification) tagged Rmt2 (189). Interactions could be demonstrated between Rmt2 and the nucleoporins Nup49, Nup57 and Nup100. These associations were confirmed by recipro-cal immunoprecipitations using TAP-tagged nucleoporins (Paper I). Nup49, Nup57, Nup100 and Nup116 all contain FG-repeats of the type GLFG (76) raising the question whether Rmt2 specifically interacts with GLFG-nucleoporins, with FG-nucleoporins in general or with other types of nu-cleoporins. Immunoprecipitations of different TAP-tagged nucleoporins showed that the FG-nucleoporins Nup1 and Nsp1 could pull-down Rmt2 while no interactions were observed between Rmt2 and the structural nu-cleoporins Nup82, Nup84, Nup133 and Nup145C (Paper II). These results provide support for a specific interaction between Rmt2 and FG-containing nucleoporins. The association with Nup1 and Nsp1, which contain FG-and FXFG repeats (76), suggests that Rmt2 interacts with FG-nucleoporins in general and not only with GLFG-nucleoporins. However, it remains to be determined if Rmt2 directly interacts with these nucleoporins or if other proteins mediate the interactions. FG-nucleoporins have been demonstrated to participate in the transport through the NPCs and to interact with transport receptors (reviewed in (74)). To address the function of Rmt2 at the NPC, we investigated nuclear trans-port mediated by NES- and NLS-signals in the rmt2Δ mutant. Preliminary results show a delay in the NES-mediated protein export (Paper II). The export delay in rmt2Δ was observed at elevated temperature suggesting that Rmt2 does not have a strong influence on transport. Taken together, the as-sociation of Rmt2 with nucleoporins actively participating in nucleocyto-plasmic shuttling and the influence of Rmt2 on nuclear export suggest a role for Rmt2 in nuclear transport. It is tempting to speculate that Rmt2 is part of a transport complex interacting with the FG-nucleoporins. Arginine methyla-tion has previously been found involved in nuclear transport. The major ar-ginine methyltransferase in yeast, Hmt1, functions both in export of mRNA-associated proteins and in the processing of transcripts (163). The previous finding that Rmt2 methylates the ribosomal protein L12 (170) prompted us to investigate whether Rmt2 is associated with ribosomes. Our result showed that Rmt2 did not fractionate with ribosomes, indicating that Rmt2 does not bind to ribosomes or interact with L12 on the ribosomes. This suggests that the methylation of L12 occurs prior to the assembly of L12 into the ribosomes. No differences were observed of the steady-state localization of L12 between wild type and rmt2Δ strain (unpublished results), indicating that Rmt2 methylation is not important for L12 localization. However, it

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cannot be excluded that Rmt2 could influence the kinetics of L12 localiza-tion or the localization during certain cellular stages. The fission yeast argin-ine methyltransferase Rmt3 methylates the ribosomal protein S1 and deletion of Rmt3 influences the ratio between the 40S and 60S ribosomal subunits (152). Analyses of the ratio between the 40S and 60S in rmt2Δ cells did not display differences compared to wild type cells (unpublished results). This observation has also been made in other studies (190). The interactions be-tween Rmt2 and L12 seem to be transient. The co-immunoprecipitation stud-ies of L12 showed that Rmt2 was not pulled-down by L12-TAP (Paper I). L12 is translated in the cytoplasm and then imported to the nucleus to be assembled with the ribosome. The function of Rmt2 methylation could be important for any of those processes, and suggests a role for Rmt2 in ribo-some biogenesis or RNA processing. Apart from the L12 protein, there are no other known substrates for Rmt2. Bioinformatic analyses in the yeast database found a number of proteins with sequences similar to the Rmt2 target site in L12 that could be potential sub-strate candidates. Among the interesting candidates were Lsm4, a RNA-processing protein, Tfc3, a subunit of RNA polymerase III, Imp3, involved in rRNA processing, Ypt52, a GTPase, and Pex2, a perxisomal membrane protein. Human Lsm4 has previously been found to contain arginine methy-lation (191) rendering Lsm4 to be an interesting substrate candidate. An association between Rmt2 and Lsm4 was manifested by immunoprecipita-tion of Lsm4-TAP showing that Rmt2 co-purifies with Lsm4 (Paper II). Further studies need to be undertaken to determine if Lsm4 is indeed a sub-strate for Rmt2 or if these two proteins interact for other reasons. Previous studies in mammalian cells have implicated essential functions for arginine methylation in processing of Sm-proteins that interact with snRNA involved in splicing (165). Lsm (Like-Sm) proteins associate with U6 snRNA and have been implicated in functions in several stages of RNA processing (re-viewed in (192)). The physical interaction with Lsm4 thus suggests a func-tion for Rmt2 in RNA processing. To investigate the impact of Rmt2 on the gene expression level, expression-profiling analyses were performed on wild type and rmt2Δ strains using DNA microarrays and real-time PCR. Only two genes were found affected in the rmt2Δ mutant compared with the wild type strain, the MYO1 gene encoding type II myosin heavy chain, and an uncharacterised ORF. This demonstrates that Rmt2 has a very little effect on transcriptional regulation (Paper I). However, Myo1 has an essential role in cytokinesis (193) sug-gesting that the function of Rmt2 could be linked to the cell cycle or active growth. Interestingly, several other genes related to the cell cycle were found affected in the microarray analyses of rmt2Δ, but these effects could not be validated by real-time PCR. These results open for a role of Rmt2 in actively growing cells. Support for this can be seen in the expression profiling studies

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presented in the yeast database revealing similar expression profiles for RMT2 and genes involved in rRNA processing, protein biosynthesis and transcription. Arginine methylation has been related to several steps in RNA processing and regulation of nucleocytoplasmic transport (reviewed in (140, 142, 146)). From the results in our studies, Rmt2 seems to be involved in cellular proc-esses similar to those found for other arginine methyltransferases. Although several processes involving arginine methylation is linked to the nuclear envelope, no arginine methyltransferase has previously been physically linked to the nuclear periphery. Several arginine methyltransferases have however been located to the nucleus and nuclear proteins are often reported as targets for arginine methylation. This posttranslational modification thus appears to be important for nuclear processes. The type IV arginine methyla-tion executed by the Rmt2 enzyme is not well characterized and the function of it has not been elucidated. The presence of Rmt2 in the nucleus and the nuclear periphery suggests this specific arginine methylation to have an im-portant function in processes connected to the nuclear border.

2.2.3 Cwh43 is an integral membrane protein of the inner nuclear membrane (paper III) Here, we have investigated the functions of the Cwh43 protein. The localiza-tion of Cwh43 to the nuclear membrane was confirmed by immunofluores-cence- and electron transmission (EM) microscopy using an antibody recog-nizing a peptide fragment in the C-terminus. The Cwh43 protein is expressed in low levels and from those experiments it appeared that Cwh43 is difficult to detect in whole cells. Upon overexpression from a GAL promoter, Cwh43 accumulates around the nucleus. In isolated nuclei, endogenously expressed Cwh43 is localized in foci of various sizes around the nucleus but does not co-localize with the NPCs. It is also located at the border of the nucleolus between the nucleolus and the DNA area. In EM analyses, Cwh43 is clearly located at the inner side of the nuclear membranes and at the nucleolus. The Cwh43 has previously been predicted to contain several transmembrane regions (135). This together with the localization data suggest that Cwh43 is an integral membrane protein of the inner nuclear membrane. The sequence analyses of Cwh43 also predicted it to contain a hydrophilic C-terminus in addition to its transmembrane regions (135). BLAST searches of the C-terminus sequence unveiled weak sequence similarities with DNA binding proteins. Secondary structure predictions of the C-terminus dis-played significant structural similarities with the human Ape1/Hap1 enzyme, which is involved in base excision repair by cleaving apurinic or apyrimid-inic (AP) sites (194). Secondary structure predictions of the C-terminus of human Cwh43 displayed a similar structure as the yeast Cwh43. These re-

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sults suggest that Cwh43 binds DNA. To test this possibility, the recombi-nant C-terminus was incubated with linearized plasmid DNA or short DNA oligos and the electrophoretic mobility of the DNA was analysed. There was a clear retention of the DNA migration on the gels in the presence of the Cwh43 C-terminus. In addition, analyses of the enzymatic activity of the recombinant C-terminus showed that it has the ability to cleave DNA with apurinic sites in the same manner as the Ape1/Hap1 enzyme (Rachel Amouroux, unpublished results). These results infer that Cwh43 interacts with DNA and the structural similarity with Ape1 suggests a possible func-tion in DNA repair. Disruption of the CWH43 gene is lethal at 39°C (135). When the mutant cells were grown in the presence of MgCl2 or CaCl2 the lethality were res-cued, indicating that Cwh43 is important for ion homeostasis. To investigate the cellular pathways in which CWH43 acts, a screen was conducted to search for genes that upon overexpression could rescue the lethality of cwh43Δ at 39°C. Two genes were identified in this study, FOB1 and RTS1. Fob1 is a nucleolar protein required for replication fork blocking and rDNA recombination by regulating the number of rDNA repeats (195, 196). Rts1 is a regulatory subunit of the protein phosphatase 2A implicated in global stress response and nutrient signalling. Recently, Rts1 was also shown to have a function in protecting centromeric sister chromatid cohesion during meiosis (197-199). This result implies that Cwh43 is genetically linked to rDNA recombination, stress response, nutrient signalling and possibly meio-sis. Since Cwh43 was found close to the nucleolus, Fob1 could be related to the nucleolar function of Cwh43. Both Fob1 and Rts1 are involved in proc-essing of DNA and could possibly be related to the DNA binding function of Cwh43.

To examine the influence of CWH43 on the transcriptional level, an expres-sion profiling analysis was performed on cw43Δ cells using DNA microar-rays. Nine genes were found down-regulated in cw43Δ cells compared with wild type cells. These results were confirmed by real-time PCR. Among the genes affected were the PDR3 transcription factor and a number of target genes for PDR3. The transcription factors Pdr1 and Pdr3 are regulating genes in the pleiotropic drug resistance (PDR)-pathway that responds to cytotoxic substances (200). The Pdr1/Pdr3 target genes are also rapidly in-duced by membrane perturbing agents, which indicate them to function in membrane lipid organization (200). The expression profiling data connect Cwh43 to the PDR-pathway and metabolism. Cwh43 has previously been related to cell wall signalling via the PKC path-way and to lipid remodelling (135, 137). Our data connect Cwh43 to DNA metabolism, the PDR-pathway, nutrient signalling and stress response. The Pkc1 protein has in addition to its well-established role in cell wall integrity

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signalling been implicated in e. g. cell cycle progression, mitotic recombina-tion and phospholipid synthesis (reviewed in (129)). Thus, our results of the Cwh43 may be connected to the role of Cwh43 in PKC signalling and Cwh43 may be an additional link between PKC pathways and nuclear func-tions. Cwh43 could have several functions. One could be a role in signalling and another a role related to DNA binding. It is also possible that these func-tions could be integrated. In higher eukaryotes Cwh43 has been split into two proteins. This may reflect that Cwh43 is involved in several functions in yeast. Interestingly, in the studies of Cwh43 in lipid remodelling it was shown that both the N-and C-terminals were important for this function (136, 137). Observations have been made that inner nuclear membrane proteins have important functions in signal transduction, transcriptional regulation and DNA repair both in yeast and mammalian cells (10, 42, 67). Cwh43 may thus be an inner nuclear membrane protein that functions both in signalling and DNA repair or DNA metabolism.

2.2.4 Myr1is a novel membrane associated protein (paper IV) In the screen for nuclear membrane associated proteins using the MEGA10 detergent we identified Ypt6, a Rab GTPase. Ypt6 has previously been re-ported to localize to Golgi (183). Our unpublished observations of myc-tagged Ypt6 in wild type cells and isolated nuclei displayed spots in the cy-toplasm and at the nuclear periphery. Preliminary results have shown that the ypt6Δ mutant displays disturbed localizations of the nucleolar protein Nop1 and the nuclear pore complexes as well as minor affects on the general nu-clear import (unpublished results). This suggests that Ypt6 could have a function in the biogenesis of nuclear membranes and/or nuclear transport. Components of the vesicular traffic have previously been found linked to the nuclear envelope. One constituent of the NPC, Sec13, cycles between the NPC and the ER (201) and vesicular traffic seems to play a role in the as-sembly of NPCs (96, 97). In addition, mutants of proteins involved in vesicular traffic (so-called sec mutants) display mislocalization of nucleolar and nucleoplasmic proteins to the cytoplasm and down-regulation of ribo-some biogenesis (202). Thus, there is a link between vesicular traffic and nuclear organization and Ypt6 could play a role in this connection.

To identify novel factors that interact with Ypt6 or are associated with the Ypt6 functions, we performed a high copy suppressor screen searching for genes that when overexpressed could rescue the lethality of the ypt6Δ mutant at 35ºC. In this screen we found a number of genes previously identified in other studies ((184, 186, 187). In addition, we found genes encoding the poly A-binding protein Pab1, the RNA-processing proteins Lsm4 and Lsm8, the palmiotyl transferase Erf2 and the uncharacterised ORF YPL105c. Our data,

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together with other studies of the ypt6Δ mutant, provide support for a func-tion for Ypt6 in RNA-processing and translation in addition to its role in membrane traffic (184, 186, 187). Ypt6 is potentially involved in all of these cellular functions or affects RNA-processing and translation through is role in membrane traffic. Since the function of the YPL105c gene had not previously been described, we continued to characterize its gene product. We demonstrated that overex-pression of YPL105c also rescued a deletion mutant of Ric1, which is the GEF for Ypt6 (183). YPL105c was subsequently named MYR1 for multi-copy suppressor of ytp6Δ and ric1Δ.

Large-scale two hybrid analyses have previously found Myr1 to interact with a number of proteins, among them two subunits of the translation initiation factor eIF3, the splicing proteins Msl5 and Mud1 and the negative regulator of translation Eap1 (175,203,204). These data suggest a function of Myr1 in splicing and translation initiation. The genetic interaction with Ypt6 and Ric1 also links Myr1 to vesicular traffic.

The amino acid sequence of Myr1 is organized into four domains. The N-terminus harbours a conserved GYF domain, which binds proline-rich motifs in a manner similar to SH3, WW and EVH1 domains. GYF domains are found in a number of proteins mediating protein-protein interactions (205). The GYF domain is preceded by a proline-rich region. A second domain of Myr1 consists of a hydrophobic region with a predicted amphipatic α-helix. A coiled-coil domain with a NLS sequence follows the hydrophobic region. The C-terminus is conserved among fungal species. In S. cerevisiae, Myr1 share homology with the Smy2 protein implicated in COPII vesicle forma-tion (206). Subcellular localization studies of myc-tagged Myr1 displayed a reticular pattern in the cytoplasm. In biochemical analyses Myr1 was demonstrated to sediment at low speeds indicative of an association with heavy cellular structures such as membranes or organelles. Myr1 could not be solubilized in the presence of detergent suggesting a non-membraneous association. In contrast, a membrane-association of Myr1 was demonstrated by membrane flotation, where Myr1 was able to float in the absence but not in presence of detergent. Taken together, these results suggest that Myr1 has a membrane association. The presence of an amphipatic α-helix in Myr1 and the sedi-mentation results suggest a peripheral interaction with membranes. The am-biguous observations of the Myr1 membrane association could be due to an interaction both with membranes and with the cytoskeleton. Yeast cells lacking the MYR1 gene do not display any significant pheno-types. However, elevated expression levels of MYR1 affect the cells. Strong

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overexpression of MYR1 generates cell death. The N-terminus containing the GYF domain was demonstrated to be responsible for the observed lethality. Cells overexpressing MYR1 from multicopy plasmids manifested uneven expression levels and disturbed DNA morphology. In addition, a deletion mutant of the nucleoporin Nup133 displaying a pore clustering phenotype (207) exhibited a normalised phenotype when overexpressing MYR1. This result suggests that Myr1 could influence membrane dynamics.

To address the cellular functions of Myr1, a transcriptional profiling analysis was made comparing gene expression in myr1Δ and wild type cells. A few genes were affected in myr1Δ indicating that Myr1 has a small general effect on gene transcription. Up-regulations of the genes encoding the tubulin bind-ing protein Num1 required for nuclear migration and the Ras GTPase activa-tor Ira2 and a down-regulation of the RNA pol III subunit TFC3 were ob-served. Interestingly, the functions of these genes can be related to our ob-servations of the Myr1 functions. The up-regulation of Num1 could be linked to the observed influence of Myr1 on nuclear morphology and the down-regulation of Tfc3 may be related to the interactions between Myr1 and translation factors (203).

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3. CONCLUDING REMARKS

The most important findings in this thesis reflect the different functions of proteins located at the nuclear periphery and the close relationship between cytoplasmic and nuclear events.

• Two new nuclear envelope-associated proteins have been identified in the yeast S. cerevisiae, the type IV arginine methyltransferase Rmt2 and the membrane protein Cwh43. These two proteins have not previously been linked to the nuclear envelope.

• Rmt2 has been found associated with proteins of the nuclear pore

complexes that contain repeats of the sequence motif FG. This type of nucleoporins has an important function in the transport through the NPC. Rmt2 has also been found to influence nuclear protein ex-port. These results implicate a direct or indirect role for Rmt2 in nu-clear transport. In addition, a link has been established between Rmt2 and the Lsm4 protein involved RNA processing.

• Cwh43 was found to be an integral inner nuclear membrane protein

able to bind DNA in vitro and genetically related to the Fob1 and Rts1 proteins involved in recombination, nutrient signalling and stress response. Gene expression profiling of the cwh43 deletion mu-tant suggests a connection to metabolism. Based on these results we hypothesize that Cwh43 functions in signal transduction pathways related to metabolism. Cwh43 also provides a novel link between PCK signalling and nuclear functions.

• A novel protein Myr1 has been identified and characterized showing

that it is related to vesicular trafficking pathways involving the Rab protein Ypt6 and the GEF of Ypt6, Ric1. Genetic and biochemical studies showed that Myr1 influences membrane dynamics, transla-tion and cell signalling.

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4. FUTURE PERSPECTIVES

Future studies should further explore the functions of the proteins described in this thesis. Further studies on the Rmt2 protein should address its function in nuclear transport and in RNA metabolism. Future experiments will reveal whether Rmt2 also affects RNA export and interacts with transport cargos. A crucial question concerning the function of Rmt2 is the potential presence of sub-strates other than the already identified L12. The Lsm4 protein is here an interesting substrate candidate. Also, the FG-nucleoporins found associated with Rmt2 could be potential substrates. The Nup57 protein contains a se-quence similar to the sequence targeted for methylation in L12 and is a po-tential substrate candidate. Several other binding partners for Rmt2 have been identified – Sit4, a protein phosphatase 2A (176), the meiotic recombi-nation protein Mer2/Rec107, the autophagy protein Atg12 and the uncharac-terised ORF YBR261c (175) and it is possible that the functions of Rmt2 are associated with those proteins. There are no true mammalian homologues to Rmt2 apart from the GAMT enzyme. It would be interesting to evaluate if there are enzymes in higher eukaryotes able to methylate the δ-nitrogen on arginines. In fact, the poly-clonal Rmt2 antibodies recognise a single protein band in HeLa extracts (unpublished observations) suggesting that there may exist a human Rmt2 homologue. Analysing mammalian cell extracts for the presence of δ-nitrogen arginine methylations would answer this question. Furthermore, the L12 protein is highly conserved among eukaryotes. Analysing mammalian L12 for arginine methylations would also give information on the existence of a mammalian Rmt2 enzyme. A possibility is that the Rmt2 arginine me-thylation has been replaced by another kind of arginine methylation in higher eukaryotes. Future studies on the Cwh43 protein should concern identifying binding partners to elucidate its function on a molecular level. Current experiments address the enzymatic activity of Cwh43 and a possible role in recombina-tion events. Further studies will also show if Cwh43 binds to DNA in vivo

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and show binding preferences to certain DNA sequences. An interesting question is also the structure of Cwh43 and its topology in the membrane. Future studies will address what function Ypt6 have at the nuclear periphery. An attractive hypothesis is that Ypt6 is involved in protein or lipid traffic between ER and the NE. Alternatively membrane biogenesis is disturbed in ypt6Δ, which affects the NE biogenesis. Regarding the Myr1 protein, future studies should address the molecular functions of Myr1. Further investigations of its binding partners and mem-brane-association would tell more about its function.

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5. SAMMANFATTNING

Proteiner associerade med de yttre och inre kärnmembranen har viktiga funktioner i en rad cellulära processer, t. ex transport in och ut ur kärnan, förankring av kromatinet i kärnmembranet, koordinering av cytoplasmiska processer och kärnprocesseroch transkriptions-reglering, DNA-reparation och signallering. Mutationer i proteiner relaterade till kärnmembranet ligger bakom ett flertal humana sjukdomar, bl a muskelsjukdomar och cancer. Kunskap om hur proteiner i kärnmembranet fungerar är därför av stor betydelse för att förstå uppkomsten av dessa sjukdomar. För få ökad kunskap om kärnmembranproteiners funktioner, genomfördes en screen för kärnmembran-associerade proteiner i bagerijäst, Saccharomyces cerevisiae. I denna studie identifierades ett argininmetyltransferas, Rmt2, och ett protein inblandat i cell signalering, Cwh43. Den här avhandlingen beskriver funktionerna hos Rmt2 och Cwh43 samt identifieringen av ett nytt protein, Myr1. Våra resultat är viktiga pusselbitar i förståelsen för hur kärnmembranet och proteintrafiken fungerar i olika cellulära processer. Rmt2 sitter associerad med kärnporproteiner innehållande FG-motiv. Dessa typer av kärnporproteiner har en viktig roll i transporten av makromolekyler genom kärnporkomplexet. Rmt2 påverkar också exporten av proteiner ut ur kärnan. Rmt2 interagerar med Lsm4 proteinet som har en roll i RNA-processning. Våra resultat tyder på att Rmt2 har en roll i kärnmembrantransport och möjligen också i RNA metabolism. Avhandligen visar också att Cwh43 är ett integralt membranprotein som sitter i det inre kärnmembranet och vid nukleolus. C-terminal av Cwh43 kan binda DNA i in vitro-studier. Genetiska studier av en deletions-mutant av Cwh43 visar på kopplingar till metabolism, DNA relaterade funktioner och signalvägar kopplade till stress och näringsupptag. Våra resultat pekar mot att Cwh43 är inblandad i flera olika cellulära processer såsom signallering och DNA metabolism. Myr1 har identifierats som en gen som vid överuttryck kan rädda deletionsmutanter av membrantrafik-proteinerna Ypt6 och Ric1. Myr1 binder membran och uppvisar en nätverkslinknande struktur i cellerna. Överuttryck av Myr1 påverkar kärnans morfologi och fördelning av kärnporer runt kärnan. Dessa resultat tyder på att Myr1 har en funktion i membran-dynamik.

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6. ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to all the people who in various ways have contributed to this thesis. My supervisor, Ann, for encouraging and supporting me through this project on its long and sinuous way. My colleagues Jean-Marc, Alexander and Arunas, for teaching me yeast and microscopy and for nice company in the lab. Rosa, the greatest friend one could ever ask for, for being such a wonderful friend, “kombo” and colleague. Thank your for all you support and for al-ways being there when I need you! Ulrika, for being an amazing friend and study partner. Thank you for all the good advices and ideas you always come up with. Karl Ekwall and Claes Gustafsson for valuable help with setting up the microarray- and TAP-tag studies and for great scientific inspiration. Anna and Maria for funny times during teaching. The people in the groups of Karl Ekwall and Anthony Wright: Ingela, Julian, Marianna, Rebecca, Carolina, Helmi, Monika, Fredrik, Yong-tao, Rachel, Stefan, Maria and Pernilla. Thanks for nice lab company, coffee times and for kindly sharing stuff and good advices. To the people of Odd Nygård´s group: Odd, Birgit, Galyna, Ivo, Gunnar, Marika, Sofia and Lovisa. Thanks for all your help with sucrose gradients and DNA assays and for kindly sharing lab equipments. All office-, lunchroom- and lab-friends at Södertörn now and in the past. All technical and administrative staff at Södertörns Högskola for invaluable help with computers, dishes, autoclaving and various other things.

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The people at the Department of Biochemistry and Biophysics at Stockholm University, especially Stefan Nordlund and Åke Wieslander, for accepting me as a PhD student at your department. My old landlady, Lena, for making me starting exercising and for scientific inspiration. My Stockholm-friends: Karmen, Jeanette, Per, Monika and Johan. Thanks for being such good friends and having patience with me at stressful times. My Gothenburg-friends: Veronica, Lena, Marie, Sara and Malin. Thank you for all the fun times during the chemistry studies, at the chorus, parties and other times. Malin and Linda, you have inspired me a lot. Mom and Dad, I can in no words express how grateful I am for your out-standing support through these years. Without your help I would never have manage this thesis. Oskar, my lovely brother, for taking me down to earth and making me see things in other perspectives. Maria, Johan, Lill-Johan, Emelie, Elsa, Tova, Hanna and Jörgen. It is great to be part of your family! And last, but not least, to Carl, my hero, for so wonderful taking care of me, for your never-ending support and coaching. You are the best. I love you!! This work was supported by grants from the Swedish Research Council, the Magnus Bergvall Foundation, the O. E. and Edla Johansson Scientific Foun-dation, the Carl Trygger Foundation for Scientific Research, the Lars Hierta Memory Foundation, Stockholms Läns Landsting and the Royal Swedish Academy of Research.

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