Beyond the central dogma

Central dogma culminates with synthesis of protein in cytoplasm

But can’t mix proteins, polysaccharides, lipids and nucleotides together and get a living cell

Formation of a cell requires the context of a pre-existing cell

Cell structures (organelles; mitochondria, chloroplasts, Golgi, ER) and organization must be inherited, just like

DNA

Epigenetics

Lecture 14 cont’d

Intro to protein import into organelles

Signal sequences

Import into the nucleus

Import into mitochondria and chloroplasts

Import into ER, vesicle trafficking

Note - in the next few lectures I will show many figures from Molecular Biology of the Cell 4th ed.

(Alberts et al.)On reserve at Marriott

Nuclear import occurs through pores in the double membrane

All nuclear transport occurs through nuclear pores

Outer nuclear membrane Inner nuclear membrane

“Nuclear envelope”=

Nuclear lamina

Perinuclear space

ER

ECB 15-7

Nuclear pores

What molecules must be imported into nucleus? Exported?

Nuclear pores are large protein complexes

Multiple copies of ~100 different proteins (nuclear pore proteins = NPPs) totaling >125 million

daltons!

Cytoplasmic face

Nuclear face ECB 15-8

Cytosol

Nucleus

Annular subunit of centralchannel or transporter

Nuclear basket or cage

Cytplasmic fibrils

Nuclear lamina

Nuclear envelope

Transport of large molecules is active - requires GTP

Small molecules (< 60 kDa), or about 9 nm diameter) enter or exit nucleus by passive diffusion

Larger molecules must be actively tranported:

(1) binding to transporter; and

(2) transport thru nuclear pore using GTP

Nuclear pores also required for active export of RNPs (including ribosome subunits, mRNA, tRNA

etc.)Import and export occur through same pores

A nuclear localization signal (NLS) is necessary and sufficient for nuclear import of proteins

The “classical” signal for nuclear import includes multiple basic amino acids (K = lysine and R = arginine)…example P-P-K-K-K-R-K-V

NLS can be anywhere in protein sequence

Simplified view of nuclear transport

(importin)

NLS(cargo)

ECB 15-9

Pore opens

GAP

Molecular “switches”

GTPase

GTP

GTPase

GDP

Pi

GDP

GTP

“on” “off”GEF

Energy for transport provided by G proteins (GTP binding proteins; large

family)

GAPGAP

GTPaseGTPase

GTPGTP

GTPaseGTPase

GDPGDP

Pi

GDP

GTP

“on” “off”

GEFGEF

GAP = GTPase Activating ProteinGAP = GTPase Activating Protein

GEF = Guanine Nucleotide Exchange FactorGEF = Guanine Nucleotide Exchange Factor

RANRANGTPase used inGTPase used in

nuclear transportnuclear transport

Q: relation to proteintransport??

Nuclear import/export cycle is driven by GTP hydrolysis

Directional protein import is driven by GTP hydrolysis

Cytoplasm

Nucleus

Importin (NLS receptor) binds cargo (with NLS) in cytoplasm

Importin-cargo transported into nucleus thru nuclear pore

Ran-GTP in nucleus binds importin, importin releases NLS (cargo)

Ran-GTP-importin exported from nucleus thru pore

Ran-GAP stimulates GTP hydrolysis in cytoplasm by Ran

Ran-GDP releases importin in cytoplasm

Ran-GDP transported into nucleus (not shown)

Ran GEF stimulates nucleotide exchange restoring Ran-GTP.

NLS

NLSRan-GDPRan-GTP

Importin

Ran-GTP

Importin

Ran-GTPImportin

NLS

Importin

NLS

Importin

Ran-GDP

+

RanGEF

GDPGTP

Ran

GAP

Pi

Specific signals direct export from the nucleus: lessons from HIV

GpppC AAAmRNA (2 kB)

Processing

Transcription

Unspliced vRNA (9 kB)

TransportTranslation

Rev

Cytoplasm

Nucleus

Human T lymphocyte

HIV

Reverse transcription

Uncoating

vRNA

DS vDNATransport

Integration

Progeny virus exits host cell by budding

Alternative splicing produces over 30 mature mRNA that are exported and translated

One protein, Rev contains a NLS and is tranported into the nucleus

Rev binds “Rev-response element” on vRNA

Rev-RNA complex exported, RNA packaged and virus leaves cell

Nuclear Export Signal Rev is req’d for export of Rev-vRNA from nucleus

Human immunodeficiency virus (HIV) is a “retrovirus:”

RNA genome with DS DNA intermediate

RNA is “reverse transcribed” to make DS DNA

Unspliced vRNA is trapped in nucleus (contains introns-no export)

Lecture 14

Intro to protein import into organelles

Import into the nucleus

Import into mitochondria and chloroplasts

Organelle DNA - encodes small % proteins, human mitochondria encode only 13 proteinsRest (thousands) encoded in nucleus, transcribed, exported to cytoplasm, translated and imported into correct organelleAnd correct compartment in that organelle

Recall mitochondrial and chloroplast structure

Translate mRNA for mitochondrial matrix protein in vitro

Proteins contain N-terminal “signal sequence”

Import into mitochondria is post-translational

Digest with protease

Protein degraded

Add “energized” mitochondria

Protein imported into mitochondria

Imported matrix protein is protected from added protease

Import into mitochondria and chloroplasts is post-translational

Trypsin

Trypsin

Positive charge (red) clustered on one face of helix…

Non-polar aa (green) on the other…

Import is directed by a signal sequence at the N-terminus of mitochondrial proteins

No conserved sequence

Predicted to form “amphipathic” a-helix

Cleaved after protein is imported

MBoC (4) figure 12-23© Garland Publishing

TOMs and TIMs: Import into the mitochondria matrix requires two membrane transporters…

Mitochondrial import signal binds receptor in outer membrane (assoc w “TOM”)

Matrix

Outer membrane

Inner membrane

Cytoplasm

Intermembrane space

Matrix protein w N-terminal signal sequence

Removal of signal sequence

Mature matrix protein

Import receptor

“TOM”

“TIM23”

See ECB figure 15-10

“Contact site” (close apposition of OM & IM)

Transport thru aqueous channels: “TOM” and “TIMs” (Translocaters in Outer/Inner Membrane)

Protein import into mitochondria requires energy…

(1) Electrical potential () across inner membrane req’d to initiate transport

Adapted from MBoC (4) figure 12-27© Garland Publishing

ATP

ADP + Pi

- - - - - - - - - - - - - -

+ + + + + + + + + + + + + + +

Cytoplasm

IMS

Matrix

Outer membrane

Inner membrane

ATP

ADP + Pi

CytosolicHSP70

MitochondrialHSP70

TIM23

TOM

(3) Mitochondrial HSP70 refolds protein after import (ATP used)

(2) Cytosolic HSP70 unfolds protein for import (ATP used)

How are proteins targeted to other mito membranes/compartments?../L14OrganelleImport/15.5-mito_import.mov

How are proteins targeted to mitochondrial membranes and compartments? …the direct route

Cytoplasm

Matrix

Inner membrane

Outer membrane

IMS

TOM

TIM“Stop transfer” signal

Protein in IMSProtein in IM

Cleaved stop transfer(degraded)

Matrix signal (cleaved and degraded)

As before, signal sequence directs import through TOM/TIM23…

Adapted from MBoC (4) figure 12-29

“Stop transfer” signal interrupts translocation through TIM23, releasing protein to inner membrane…

Cleavage of stop transfer signal releases protein to intermembrane space…

Import into the thylakoid requires multiple signals

A “transit peptide” (an amphipathic helix) targets to chloroplast stroma

(similar to mitochondrial signal peptide, but NOT interchangeable!)

Evidence for four paths to thylakoid Adapted from MBoC (4) figure 12-30 © Garland Publishing

Receptor

Thylakoidsignal

Transporters

Transit peptide cleaved

Transitpeptide

Outer membrane

Inner membrane

Stroma

IMS

Thylakoid

Cytosol

Protein targetingV

esi

cle t

arg

eti

ng

Secretory vesicles

Lysosomes

Endosomes

RetrievalTransport

RER

Golgi

Plasma membrane

See ECB figure 15-5

NLS: (basic)

NES: (L-rich) Signal peptideCytoplasm

Pro

tein

ta

rgeti

ng

Nucleus Mitochondria

Chloroplasts

Additional signals for subcompartments

Next two lectures

L15: Protein and vesicle targeting

ECB 15-5

Today - import into ER, begin vesicle targeting

GFP-protein in plant cell ER

ER network is extensive

TEM of RER in dog pancreasNote ribosomes on membrane

GFP-protein in plant cell ER

Vesicles derived from ER by biochemical prep are termed microsomes

Some ribosomes bind to ER

What is the evidence for cotranslational transport?

ECB 15-12

Transport of protein into ER is cotranslationalAdd RER microsomes AFTER translation…

Product still ~2kDa larger than in vivo product…Add protease…Product degraded…

Add RER microsomes DURING

translation…

Product processed to mature form…Add protease…Product protected…

INSIDE microsomes!

In vitro product ~2 kDa larger than in vivo product~15-25 addtnl aa at N-terminusAdd protease - product degraded

Translate mRNA in vitro…

The “Signal Hypothesis”

1. The signal for translocation of a secretory protein into the ER resides in the nascent polypeptide, in the form of a leader “pre-” sequence or “signal peptide;”

2. Translocation of the polypeptide across the ER membrane is co-translational (unlike import into nucleus, mito, and chl); and

3. the signal peptide is cleaved post-translationally in the ER lumen by a “signal peptidase.”

From results of experiments such as these, Dobberstein and Blobel proposed a hypothesis

Blobel - Nobel prize 1999

ER Signal Sequence

No conserved sequence

Signal sequence is 12-25 amino acids Predicted to form-helix with hydrophobic core (yellow aa above)

Signal sequence is both necessary and sufficient for import into ER

Necessary

Sufficient

Requirements for targeting and translocation into the ER

1. “Signal sequence” : hydrophobic a-helix in nascent protein

2. “Signal recognition particle (SRP):” cytoplasmic complex of protein and RNA binds signal sequence

3. “SRP-receptor:” integral ER membrane protein

4. “Translocon:” an aqueous channel through ER membrane (sec61 complex)

Targeting to RER

1. Translation exposes signal sequence outside ribosome

2. SRP -a complex of 300bp RNA and 6 proteins- binds the signal sequence in nascent protein, transiently arrests translation

3. SRP-arrested ribosome binds SRP receptor in ER membrane (targeting)

4. Ribosome and polypeptide handed to a translocation channel (“translocon”). SRP and SRP-R are recycled (requires GTP hydrolysis). Translation resumes and translocation begins

ECB 15-13

Proteins destined for secretion enter ER lumen

Signal peptide targets nascent protein to RER as before

Signal peptide is cleaved by signal peptidase associated with translocation channelTranslation and translocation are completed, releasing completed polypeptide into lumen of RERSignal peptide is degraded What about membrane proteins??

ECB 15-14

As before, signal peptide targets nascent protein to RER

However, “Stop transfer” sequence halts translocation

Membrane proteins contain stop transfer sequence

Protein is released from translocon

Stop transfer sequence acts as transmembrane domain

ECB 15-15

Double- and multipass membrane proteins

Internal signal sequence targets nascent protein to RER…

“Stop transfer” sequence halts translocation and releases protein from translocon… Signal sequence and stop transfer sequence act as transmembrane domains

ECB 15-16

Protein folding in the ER is assisted by “BiP”…“Binding protein” (HSP70 family of ATPases) in ER lumen binds nascent polypeptide as it is being translocated, and assists folding (and translocation?)…

BiP binds nascent protein during translation/translocation…

RER membrane

ER Lumen

Translocon(sec 61 complex)

Signal peptide

Signalpeptidase

“Secreted protein” in

lumen of RER

Adapted from MBoC (4) figure 12-46.See ECB figure 15-14

N

N

C

N

C

Signal peptide

BiP

BiPBiP

BiP

BiP

ADP+Pi

ATP

Release of BiP from folded polypeptide requires energy (ATP)…

Incorrectly folded proteins are held in ER until folded properly, or are targeted for degradation…

The topology of a membrane protein can be predicted…

Hydrophobic -helices of 15-25 aa are predicted to be membrane spanning domains…and also function as “topogenic sequences.” Seven domains in rhodopsin

“Start transfer” initiate protein translocation, “Stop transfer” sequences halt translocation…

Note start sequences can be in either orientation

Adapted from MBoC (4) Figure 12-50 © Garland Publishing

H2N- -COOH

1 2 3 4 5 6 7

Topology of Rhodopsin

COOH

NH2

ER Lumen

Cytoplasm

1 2 3 4 5 6 7

Hyd

roph

ilic

Hyd

roph

obic

200100

Hydropathy plot for Rhodopsin

Start

6

Stop

71

Start

2

Start Stop

3

Start

4

Stop

5

A B C D

A B C D

Review of the “Signal Hypothesis”

1. The signal for translocation/insertion of a protein into the ER membrane resides in the nascent polypeptide, in the form of a “signal sequence.”

2. Translocation of the polypeptide across the ER membrane is co-translational

3. The signal peptide (of secreted proteins) is cleaved post-translationally in the ER lumen by a “signal peptidase.”

4. Four components: (1) signal sequence, (2) SRP, (3) SRP-R, and (4) translocon

5. Uncleaved signal sequences (and “stop transfer” sequences) function as transmembrane domains in integral membrane proteins…

6. The topology of a protein can be predicted from the “hydropathy” plot of its amino acid sequence… 15.7-ERprotein_trans.mov

Vesicle targeting

Protein and vesicle targetingV

esi

cle t

arg

eti

ng

Secretory vesicles

Lysosomes

Endosomes

RetrievalTransport

RER

Golgi

Plasma membrane

See ECB figure 15-5

NLS: (basic)

NES: (L-rich) Signal peptideCytoplasm

Pro

tein

ta

rgeti

ng

Nucleus Mitochondria

Chloroplasts

Additional signals for subcompartments…

15.1-cell_compartments.mov

Membrane cycling

Secreted proteinsPlasma membrane proteinsExocytosis

(secretion)

endocytosis

ECB15-17

Transport is highly regulated so vesicles carry appropriate cargo for their specific destination

Lumen of organelle is equivalent to outside of cell

xx

xx

x

x

What about membrane protein in ER?

Begin with ER to Golgi

Modification of proteins begins in ER

ECB 15-22

Disulfide bridges

Glycosylation - common in plasma membrane and secreted proteins

Most common glycosylation is addition of a specific oligosaccharide (14mer) to asparagine during translation. Addition is to the NH2 group; N-linked glycoproteins

Addition is done in a single step by transfer from specialized dolichol lipid

This oligo is then extensively modified in diverse ways

Modification begins in ER: Transported to Golgi for more processing

Asn-X-Ser

MBoC (4) figure 13-22 © Garland Publishing

From the ER, proteins are transported to the Golgi

Vesicular-tubular clusters to CGN

RER

Nuclear envelope

Proteins leave the ER in transport vesicles budding from exit sites…

Transport vesicles from ER fuse to form vesicular-tubular clusters…

Vesicular-tubular clusters enter the Golgi by fusing with the cis-Golgi network (CGN)

Glycoproteins are “processed” as they pass thru the Golgi…

Golgi

ECB 15-24

From the ER, proteins are transported to the Golgi

cis-Golgi network (CGN)

cis

trans

medial

Vesicular-tubular clusters in from RER…

Proteins leave the ER in transport vesicles budding from exit sites…

Transport vesicles from ER fuse to form vesicular-tubular clusters…

Vesicular-tubular clusters enter the Golgi by fusing with the cis-Golgi network (CGN)…

Glycoproteins are “processed” as they pass thru the Golgi…

Trans Golgi network (TGN)

The Golgi is biochemically compartmentalized…

Osmium (cis)

Nucleotide diphosphatase (trans)

Acid phosphatase (TGN)

MBoC (4) figure 13-28© Garland Publishing

Glycoproteins are further processed in the Golgi

GlcNAc = N-acetylglucosamine

Mannose

Glucose

Fucose

Galactose, etc.trans

Plasma membrane

Protein synthesis

Golg

i appara

tus cis

Secretory vesicles

ER

medial

Glycosylation at H3N+…

XXNXSXX…COO-

LysosomeConstituitive secretion(Default?)

Regulated secretion

Proteins are sortedin the TGN…Constitutive secretion…Regulated secretion…Lysosome…

TGN

CGNAs protein moves through Golgi, monosaccharides are added or removed in specific Golgi compartmentsRemoval of mannose

Addition of GlcNAc

Addition of galactose

Why are membrane/secreted proteins glycosylated?

Structure and folding?

Protection of cell (protein) from external proteases?

Function?adhesion…signaling…

The plasma membrane of many (most?) cells is coated with glycoproteins

ECB 15-24

Transport through Golgicis-Golgi

network (CGN)

cis

trans-Golgi network (TGN)

trans

medial

Transport vesicles

Cisternal maturation and vesicle transport probably both contribute to membrane flow through Golgi

“Budding”

“Fusion”

2. “Cisternal maturation”

Vesicular-tubular clusters in from RER…

Transport vesicles out

1. Vesicle transport

Next time

Vesicle transport from ER to Golgi

Transport from Golgi Constitutive secretionRegulated secretionTo lysosome