Lecture 9 - endomembrane system part 4 Flashcards
What are the three fates of proteins synthesized in the ER?
retained in ER
- remains localized in RER
- move laterally through ER lumen or membrane bilayer to another ER subdomain
exit from ER
- move to ER subdomain involved in formation of transport vesicles destined for golgi - ER exit sites (ERES)
What are ERES?
distinct subdomain of ER, usually located next to cis face of golgi complex
What are the two functions of the ERES?
ERES enriched with molecular machinery responsible for formation of membrane bound transport vesicles destined for golgi
proper packing of vesicles with correct lumenal and membrane cargo proteins destined for golgi - resident ER proteins prevented from entering vesicle
Is protein trafficking throughout the endomembrane system selective?
yes
What are the distinct ‘look’ of vesicles?
small and fuzzy surface due to layer of soluble coat proteins (COPS) attached to outside
Where do COPs assemble?
on outside cytoplasmic surface of ERES membrane
What are the two functions of COPs?
- mediate membrane curvature and formation of vesicle
- recognize and concentrate specific cargo components into vesicle
What are the three major classes of coat proteins?
- COPII - move forward (anterograde transport) from ERES to Golgi
- COPI - move backward (retrograde transport) from golgi to ER and backward within golgi
- Clathrin - move from golgi to endosomes or from plasma membrane to endosomes
What are the steps of COPII assmebly at ERES?
- soluble COPII component Sar1 (GTPase) recruited from cytoplasm to ERES membrane via binding to Sec12
- sar1 binding to GTP causes conformational change which exposes the sar1 n-term
- sar1-GTP integrated into cytoplasmic facing leaflet of ERES membrane bilayer
- sar1-GTP binds several other COPII proteins (sec23 and sec24) from cytosol to ERES (form ternary complex w sec1)
promote outward bending of ERES membrane - sec24 selects vesicle proteins mediated by ER export sorting signal, binds to cytoplasmic facing domains of several types of ER integral transmembrane proteins
- all sec24 bound proteins concentrated within growing COPII protein coated vesicle
- sec23 and 24 recruit additional soluble COPII components from cytoplasm to surface of growing vesicle
- sec13 and 31 self assemble into outer, cage like lattice and act as structural outer scaffolding for vesicle
(promotes additional bending) - nascent COPII vesicle released from ERES membrane into cytosol
- after release, sec 23 promotes hydrolysis of GTP in sar1-GTP -> sar1-GDP
- GTP hydrolysis by sar1 results in disassembly of COPII protein coat - sar1GDP and other soluble COPII proteins released into cytoplasm - vescile traffics from ERES to cis golgi network (CGN) and fuses
What is sec12?
ER integral membrane protein functions as GEF that catalyzes exchange of GDP for GTP on sar1
what is sec23/24?
structural scaffolding and promote initial outward bending (towards cytosol) of ERES membrane
sec24 also involved in vesicle protein selection
What are three cargo proteins that will be included in vesicle?
membrane cargo proteins
membrane cargo-receptor proteins - bind to soluble lumenal cargo proteins
membrane trafficking proteins - required for subsequent trafficking and docking nascent vesicle with proper acceptor membrane
What is an ER export sorting signal?
located in cytoplasmic facing domains of sec24 selected vesicle membrane proteins mediates selection of vesicle membrane proteins with sec24
What does the CGN consist of?
interconnected network of vesicles and tubules located on cis face side of golgi complex
How does the COPII vesicle move across the membrane to the cis golgi?
cytoskeleton highways and molecular motors
How does a transport vesicle recognize but fuse with proper acceptor membrane steps?
- vesicle docking with acceptor target membrane
- mediated by Rab proteins
- activated Rab-GTP binds to specific Rab effector proteins on target membrane - molecular bridge
- Rabs and rab effector binding conveys vesicle targeting specific and docking - assembly of SNARE complexes
- after docking of vesicle, SNARE protein interaction brings membranes close together for fusion - through SNARE motif - Membrane fusion
- SNARE complex formation leads to fusion of membrane - Disassembly of SNARE complexes
- after vesicle fusion, SNARE complex and Rab dissociate - disassembly of SNARE mediated by NSF and SNAP
What are rabs?
large family of lipid membrane-anchored GTP binding proteins associated with all transport vesicles, key regulators of vesicle trafficking and fusion
What are SNARES?
proteins located on all transport vesicles and all target membranes, unique SNARES are associated with different membranes (specificity)
What are the two main classes of SNARES?
- v-SNARES - found on transport vesicle (v) membranes
incorporated into vesicle membrane at site of budding on donor membrane compartment - t-SNARES - found on target (t) accept membranes
What do all SNARES possess?
a SNARE motif - cytoplasmic facing coiled coil domain in but v and t SNARES that extend from vesicle target membrane surface
interact to form a stable SNARE complex and pull the vesicle and target membrane closer together
What are NSF and SNAP?
cytosolic proteins
bind SNARE complexes and unwind SNARE domains
What is the disassembly of the SNARE complex mediated by?
NSF and SNAP
What happens once the vesicle releases contents into golgi?
either remain in CGN or move to other compartments in endomembrane system
What is the fate of vesicle specific proteins or proteins that escape from ER?
return from CGN back to ER (retrograde) by specific ER retrieval sorting signals
What do most resident soluble ER proteins possess?
c-terminal KDEL sequence - serves as ER retrieval sorting signal
How are escaped ER proteins brought back to golgi steps?
- integral transmembrane protein binds to KDEL sequence of escaped ER protein 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 to ER
- KDEL receptor releases resident soluble ER protein into ER lumen
- free KDEL receptor in ER membrane returns to CGN via COPII coated ERES vesicles
What is KDEL receptor sensitive to?
higher pH in ER lumen results in conformational change and release of soluble ER cargo protein into ER lumen
What does the KDEL receptor have?
di-acidic ER export sorting signal recognized by sec24, incorporated into COPII vesicle
What do most resident ER membrane proteins possess?
cytoplasmic facing, C-term dilysine sequence - serves as ER retrieval sorting signal
this sequence on escaped ER membrane proteins at CGN is recognized by COPI
