Endomembrane System Part 4

Question 1

What is the morphology of vesicles that form at the ERES?

Answer 1

Vesicles that form at the ERES have a distinct morphology
Small (20 – 50 nm diameter) and ‘fuzzy’ surface appearance (based on EM), which is due to a layer of soluble coat proteins (COPs) attached to the cytoplasmic (outside) surface of the nascent vesicle membrane

Question 2

What two functions do COPs serve?

Answer 2

1) mediate membrane curvature and formation of a budding vesicle
2) recognize and concentrate (‘package’) specific ‘cargo’ components to be incorporated into the budding vesicle
- —> 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 with proper acceptor membrane

Question 3

Where do COPs assemble?

Answer 3

COPs assemble on the cytoplasmic surface of the ERES membrane

Question 4

What are Coat proteins?

Answer 4

Three major classes of coat proteins
Involved in the formation of distinct transport vesicles in the endomembrane system
COPII, COPI, Clathrin

Question 5

What happens to COPII coated vesicles?

Answer 5

Coated vesicles move ‘forwards’ (anterograde transport) from the ERES to the Golgi

Question 6

What happens to COPI coated vesicles?

Answer 6

Coated vesicles move ‘backwards’ (retrograde transport) from the Golgi to ER and ‘backwards’ within the Golgi

Question 7

What happens to Clathrin-coated vesicles?

Answer 7

Clathrin-coated vesicles move from the Golgi to endosomes or from the plasma membrane to endosomes

Question 8

What do Coat Proteins (COPII, COPII, Clathrin) consist of?

Answer 8

Consist of various proteins that assemble (sequentially) to form a ‘coat’ or curved ‘cage-like lattice’ on the surface of a nascent transport vesicle

Question 9

What do the ‘cage-like lattices’ on the surface of a nascent transport vesicle do?

Answer 9

Mediate membrane curvature and vesicle budding
AND
Help to select vesicle ‘cargo’

Question 10

What are the steps of COPII-coated vesicle assembly at the ERES?

Answer 10

1) Soluble COPII component Sar1(GTPase – binds GDP to GTP) is recruited from the cytoplasm to ERES membrane via its binding to Sec12
2) Sar1-GTP binds (recruits) several other COPII coat proteins from the cytosol to ER membrane surface
- -> Sar1 initially recruits Sec23 and Sec24
- -> Soluble proteins form a ternary complex with Sar1 at the ER membrane surface
- -> Act as a structural ‘scaffolding’ and begin to promote outward (i.e., towards cytosol) bending of ER membrane
- -> Beginning of COPII vesicle ‘bud’ formation
- -> Sec24 also involved in vesicle ‘cargo’ protein selection
- -> Sec24 binds to cytoplasmic-facing domains of various selected ER integral (trans) membrane proteins:
- -> Membrane ‘cargo’ proteins – destined to exit ER from Golgi
- -> Membrane ‘cargo-receptor’ proteins – bind (via luminal facing domains) to soluble (luminal) ‘cargo’ proteins destined to exit ER for the Golgi –> interact with sec24
- -> Membrane ‘trafficking’ proteins – (e.g. v- snares) – required for subsequent trafficking and docking of the nascent vesicle with proper ‘acceptor’ membrane (i.e. Golgi)
- ->Selection (recognition) of vesicle membrane cargo by Sec24 mediated by an ER export sorting signal
- -> Most common is di-acidic ER export signal (-Asp-X-Glu)
- —-> Located in cytoplasmic-facing domains of Sec24 – selected membrane proteins
- –> ER export sorting signals not found on ER resident proteins
- —–> Various other sorting signals (sequences) responsible for proper localization of proteins in the endomembrane system
- —-> All Sec24 bound proteins (and soluble proteins bound by membrane ‘cargo’ receptor proteins) concentrated within growing, COPII protein-coated vesicle ‘bud’
- —> Sec23 and Sec24 also recruit additional soluble COPII components from the cytoplasm to the surface of the growing vesicle bud
- —> Sec13 and Sec31: self-assemble into the outer, cage-like lattice and act as structural ‘outer scaffolding’ for growing COPII vesicle bud
- —-> Promotes additional outward (i.e., towards cytosol) bending of the ER membrane
- —> Eventually, nascent COPII vesicle released from the ER (ERES) membrane into the cytosol
3) After the release of nascent COPII vesicle from ‘donor’ membrane (i.e., ER/ERES), Sec23 promotes hydrolysis of GTP by Sar1 (GTPase)
- —->Sar1-GTP converted to Sar1-GDP
4) GTP-hydrolysis by Sar1 results in disassembly of COPII protein coat
- —-> Sar1-GDP and all other COPII proteins released into the cytoplasm for additional rounds of COPII- coat assembly at ERES
- —–> Results in nascent, uncoated vesicle

Question 11

What is Sec12?

Answer 11

Sec12 is ER integral membrane protein – functions as a guanosine-exchange factor (GEF) that catalyzes the exchange of GDP for GTP on Sar1

Question 12

What is Sar1?

Answer 12

Sar1 binding to GTP (Sar1-GTP) causes conformational change – expose Sar1 amphipathic hydrophobic N-terminus (serves as ER membrane ‘anchor’)
Sar1- GTP integrated into outer (cytoplasmic-facing) leaflet of ER membrane bilayer

Question 13

What is anterograde vesicle transport from the ERES to the cis-Golgi network?

Answer 13

1) Nascent ERES-derived vesicle contains soluble and membrane protein cargo and molecular machinery (e.g., Rabs & SNAREs) required for trafficking to and docking with proper ‘acceptor’ membrane
2) Vesicle traffics from ER to cis-Golgi network (GCN)
- –> nascent vesicles fuse with one another to form the GCN

Question 14

What does the GCN consist of?

Answer 14

An interconnected network of vesicles and tubules located on the cis face (side) of the Golgi complex (often located immediately adjacent to the ERES)

Question 15

How do the vesicles move in anterograde vesicle transport from the ERES to GCN?

Answer 15

Movement of vesicles through the cytoplasm, including ERES-derived vesicles moving to the Golgi (GCN), is mediated by cytoskeleton ‘highways’ and molecular motors
—-> ERES-derived vesicles to microtubules and move via kinesin motor proteins

Question 16

How does a transport vesicle recognize and fuse with the proper ‘acceptor’ membrane/organelle?

Answer 16

ERES-derived vesicle and GCN, but applies to all vesicle fusion events in the endomembrane system
Involves four main steps (Applies to COPI and Clathrin as well)

Question 17

How does a transport vesicle recognize and fuse with the proper ‘acceptor’ membrane/organelle? (Steps)

Answer 17

1) Vesicle docking with ‘acceptor’ (target) membrane
Mediated by Rab proteins
–> Large family of lipid-anchored, GTP-binding proteins associated with all transport vesicles, which are the key regulators of vesicle trafficking and fusion
‘Activated’ Rab (Rab-GTP) binds to a specific Rab effector protein on the appropriate target membrane (GCN)
—> Unique Rabs associate with different Rab effectors on different membranes – serve as a “molecular bridge”
—-> Rab effectors are sometimes cytoskeleton molecular motor proteins - enable vesicle to move on the cytoskeleton
Rabs and Rab effector binding conveys vesicle targeting specificity and docking

2) Assembly of SNARE complexes
After docking of the vesicle, SNARE protein interaction brings the membrane close together for fusion
Unique SNAREs are associated with different membranes
—-> Mediate fusion of vesicle/target membranes and provide vesicle targeting specificity (in addition to targeting specificity provided by Rab and Rab effectors)

3) Membrane Fusion
SNARE complex formation leads to membrane fusion
–> Underlying molecular mechanism for membrane fusion is not well understood

4) Dissasembly of SNARE complexes
After vesicle/target membrane fusion, SNARE complexes (and Rab/Rab-effector) dissociate (recycled for additional fusion events)
Disassembly of SNARE complex mediated by two cytosolic proteins – NSF and SNAP
—> Bind SNARE complexes x and (via ATP hydrolysis) unwind SNARE domains linking v/t- SNARES

Question 18

Explain SNARE proteins

Answer 18

A large family of integral membrane-bound proteins located on all transport vesicles and all target membranes
SNAREs actually the ones that fuse vesicle to target membrane, SNARE on vesicle and on the target membrane

Question 19

What is a SNARE motif?

Answer 19

All SNAREs contain this
Cytoplasmic-facing, coiled-coil domain in v/t-SNAREs that extend from the vesicle/target membrane surface
SNARE motifs in cognate v-SNARE and t-SNAREs interact to form a stable SNARE complex
Pulls vesicle and target membrane close together (t/v cytoplasmic domains coil together)

Question 20

What does vesicle target membrane fusion result in?

Answer 20

Vesicle membrane proteins, including membrane ‘cargo’ proteins, membrane ‘cargo-receptor’ proteins, and membrane trafficking proteins (i.e., ‘free’ SNAREs) move laterally into the target ‘acceptor’ membrane (GCN)
Vesicle soluble ‘cargo’ proteins are released from membrane receptor proteins into the interior (lumen) of GCN due to differences in pH compared to ER lumen (lower pH in GCN)

Question 21

What are the two classes of the SNARE proteins?

Answer 21

1) v-SNAREs – found on transport vesicle (v) membranes
Incorporated into the vesicle membrane at the site of budding on the ‘donor’ compartment (e.g., at the ERES, v-SNAREs are specifically incorporated (via Sec24) binding to the cytoplasmic domain of v-SNARE into the Golgi-destined vesicle membrane

2) t-SNARES – found on target(t) ‘acceptor’ membranes
e.g., at the cis-Golgi network
v-SNARE recognizes the specific t-SNARE on the membrane

Question 22

What is the fate of a newly-delivered vesicle ‘cargo’ proteins at the GCN?

Answer 22

Soluble and membrane ‘cargo’ proteins from the ER either remain in Golgi or move onto other compartments in the endomembrane systems

Question 23

What is the fate of the vesicle specific proteins or proteins that ‘escape’ from the ER?

Answer 23

Most ER-resident proteins are retained in the ER by being excluded from budding COPII transport vesicles at the ERES (do not possess an ER export sorting signal)
“Escaped” ER-resident proteins are returned from the GCN back to the ER (retrograde transport) (Golgi –> ER) by specific ER retrieval signals

example: v- SNAREs unique to ERES-derived vesicles or reticuloplasmins (BiP, calnexin)

Question 24

What do most resident soluble ER proteins process?

Answer 24

Possess a C-terminal KDEL sequence

–> Serves as ER retrieval sorting signal

Question 25

What does the KDEL receptor recognize?

Answer 25

‘Escaped’ soluble ER proteins (via COPII vesicles) in the GCN lumen recognized by KDEL receptor

Question 26

What are the steps of GCN to ER retrograde transport of ‘escaped’ proteins?

Answer 26

Integral transmembrane protein – luminal-facing domain in KDEL receptor binds to the -KDEL sequence of ‘escaped’ soluble ER proteins in GCN lumen
The cytoplasmic-facing domain of KDEL receptor (and in any ‘escaped’ resident ER membrane proteins) binds to COPI protein coat
COPI protein coat (like COPII at ERES) mediates the formation of transport vesicle at GCN (and elsewhere in Golgi complex)
COPI bind to KDEL receptor on the cytoplasmic domain side
After COPI-coat disassembly, nascent vesicle targets to and docks/fuses with ER
Soluble ER protein -KDEL receptor complexes returned back (retrograde transport) to ER
KDEL receptor ‘releases’ resident soluble ER protein into the ER lumen
Release of soluble ER protein mediated by the difference in pH in ER and GCN lumens
KDEL receptor binding sensitive to higher pH in ER lumen – results in conformation change and release of ‘cargo’
‘Free’ KDEL receptor returns to the GCN via COPII- coated vesicles
KDEL receptor has di-acidic ER export sorting signal (recognized by Sec24)
Most resident ER membrane proteins (e.g., calnexin) possess a cytoplasmic-facing C-terminal dilysine (-KKxx) sequence
Serves as ER retrieval (c-terminus) sorting signal: -KKxx sequence on ‘escaped’ ER membrane proteins at GCN recognized by COPI machinery
Proteins that cycle between the ER and GCN, 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 GCN and target to (and dock/fuse with) the ER
•Resident ER membrane proteins (and other proteins that recycle between ER and GCN) returned back to ER – retrograde transport