Lecture 9 Flashcards
What is the feet of newly synthesize proteins at the ER?
nascent, properly-folded/assembled and glycosylated soluble and membrane proteins at RER are either…
- retained in ER
remains localized in RER or move (diffuses) laterally through ER lumen or membrane bilayer to another ER subdomain (e.g., SER, nuclear envelope, etc) - exit from ER (within minutes after synthesis/folding) move to ER subdomain involved in formation of transport vesicles destined for Golgi - ER exit sites (ERES)
ERES are enriched with molecular machinery, responsible for…
1.formation (‘budding’) of membrane-bound transport vesicles destined for Golgi
2.proper packing of vesicles with correct lumenal and membrane ‘cargo’ proteins (& lipids) destined for Golgi
- resident ER proteins (e.g., BiP) prevented (not always) from entering Golgi-destined transport vesicles
Two main functions of COPs
- mediate membrane curvature & formation of vesicle
- recognize & concentrate specific ‘cargo’ components into vesicle
e.g., soluble and membrane protein (and lipids) ‘cargo’ destined for ‘acceptor’ compartment (e.g., Golgi) and molecular machinery (e.g, Rabs & SNAREs) required to traffic and dock/fuse vesicle with proper acceptor membrane
Three major classes of coach proteins involved in the formation of transport vesicles in the end membrane system
- COPII-coated vesicles move ‘forward’
(anterograde transport) from ERES to Golgi - COPI
COPI-coated vesicles move ‘backward’ (retrograde transport) from Golgi to ER and ‘backward’ within Golgi - Clathrin-coated vesicles move from Golgi Golgi to endosomes or from plasma membrane to endosomes
- COPII, COPI and clathrin assemble (sequentially) to form ’coat’ or curved ‘cage-like lattice’ (i.e., scaffolding) on surface of nascent transport vesicle to i) mediate membrane curvature and vesicle budding AND ii) help select proper vesicle ‘cargo’
COPII-coated vesicle assembly at ERES (Step 1)
Step 1:
* soluble COPII component Sar1 (GTPase) recruited from cytoplasm to ER (ERES) membrane via binding to Sec12
Sec12 - ER integral membrane protein - functions as guanosine-exchange factor (GEF) that catalyzes exchange of GDP for GTP on Sar1
* Sar1 binding to GTP (Sar1-GTP) causes conformational change – exposes Sar1 amphipathic hydrophobic N-terminus (serves as ERES membrane ‘anchor’)
* Sar1-GTP integrated into cytoplasmic-facing leaflet of ERES membrane bilayer
COPII-coated vesicle assembly at ERES (Step 2)
Step 2
*Sec24 also involved in vesicle protein selection binding to cytoplasmic-facing domains of several types of ER integral (trans) membrane proteins:
- membrane ’cargo’ proteins – destined to exit ERES for Golgi
- membrane ‘cargo-receptor’ proteins – bind (via lumenal-facing domains) to soluble lumenal ‘cargo’ proteins exiting ERES for Golgi
- membrane ‘trafficking’ proteins – required for subsequent trafficking and docking nascent vesicle with proper ‘acceptor’ membrane
*selection (recognition) of vesicle membrane proteins by Sec24 mediated by ER export sorting signal di-acidic ER export signal (-Asp-X-Glu-)
*located in cytoplasmic-facing domains of Sec24- selected vesicle membrane proteins
*all Sec24-bound proteins (and soluble proteins bound by membrane cargo-receptor proteins ) concentrated within growing, COPII protein-coated vesicle
*Sec23 and Sec24 recruit additional soluble COPII components from cytoplasm to surface of growing vesicle
*Sec13 and Sec31 - self-assemble into outer, cage-like lattice and act as structural ‘outer scaffolding’ for growing COPII vesicle bud
*promotes additional outward bending (i.e., towards cytosol) of ERES membrane
• eventually… nascent COPII vesicle released (scission) from ERES membrane into cytosol
COPII-coated vesicle assembly at ERES (Step 3 and 4)
Step 3:
*after release of nascent COPII vesicle from ERES membrane, Sec23 promotes hydrolysis of GTP in Sar1-GFP
*Sar1-GTP converted to Sar1-GDP through GAP
Step 4:
*GTP-hydrolysis by Sar1 results in disassembly of COPII protein coat Sar1-GDP and all other soluble COPII proteins released into cytoplasm for additional rounds of
*COPII-coat assembly at ERES= results in nascent, uncoated vesicle…
What is the fee of newly delivered vesicle cargo proteins at the Cis Golgi network?
soluble and membrane ‘cargo’ proteins from ER either remain in CGN or move to other compartments in endomembrane syste
What is the fate of vesicle-specific proteins or proteins that ‘escape’ from ER?
- most ER resident proteins retained in ER by exclusion from budding
COPII transport vesicles at ERES
(e.g., do not posses di-acidic ER export sorting signal) - ‘escaped’ ER resident proteins returned from CGN back to ER (retrograde transport)
by specific ER retrieval sorting signals
cis-Golgi network-to-ER retrograde transport of ‘escaped’ ER proteins
- most resident soluble ER proteins (e.g, BiP) possess ____
C-terminal –KDEL sequence
serves as ER retrieval sorting signal
cis-Golgi network-to-ER retrograde transport of ‘escaped’ ER proteins
- ‘escaped’ soluble ER proteins (via COPII vesicles) in CGN lumen recognized by KDEL receptor
- integral transmembrane protein - lumenal-facing domain binds to -KDEL sequence of ‘escaped’ soluble ER proteins in CGN lumen
- cytoplasmic-facing domain of KDEL receptor recognized by COPI protein coat
- COPI coat mediates formation
of transport vesicle at CGN - after COPI-coat disassembly, nascent vesicle targets to and docks/fuses with ER
- soluble ER protein-KDEL receptor complexes traffic back (retrograde transport) to ER
- KDEL receptor ‘releases’ resident soluble ER protein into ER lumen
- KDEL receptor binding sensitive to higher pH in ER lumen (compared to CGN lumen) – results in conformation change and ‘release’ of soluble ER ‘cargo’ protein in ER lumen
- ‘free’ KDEL receptor in ER membrane returns to CGN via COPII-coated ERES vesicles
KDEL receptor has di-acidic ER export sorting signal (recognized by Sec24)
cis-Golgi network-to-ER retrograde transport of ‘escaped’ ER proteins
most resident ER membrane proteins (e.g., calnexin) possess ___
most resident ER membrane proteins possess cytoplasmic-facing, C-terminal dilysine (–KKxx) sequence - serves as ER retrieval sorting signal
- -KKxx sequence on ‘escaped’ ER membrane proteins at CGN recognized by COPI
- proteins that cycle between ER and CGN, including KDEL receptor, membrane ‘cargo-receptor’ proteins, ER membrane ‘trafficking’ proteins (e.g, v-SNAREs) also contain –KKxx ER retrieval signal
- nascent COPI-coated vesicles bud off CGN and target/dock/fuse with ER
- resident ER membrane proteins (and other proteins that recycle between ER and CGN) traffic back to ER
(retrograde transport)
