Membrane Trafficking

Question 1

ER-targeting

Answer 1

Srp54 recognises hydrophobic N-terminal signal sequence, halt translation

SRP docks on receptor, transfers protein through Sec61 translocon

GTP hydrolysis by SRP occurs after transfer

Question 2

Folding and processing of soluble proteins in the ER

Answer 2

Signal peptidase in ER lumen, cleaves at AXA

BiP and chaperones facilitate folding

PDI mediates S-S bond formation

Glycosylated by N-linked oligosaccharide

Question 3

Transmembrane proteins ER-targeting

Answer 3

Uses an uncleavable hydrophobic signal anchors that form part of the transmembrane a-helix - mediates lateral release

Positive residues stay on cytosolic side - determines orientation

Multi-pass proteins use the first a-helix as the anchor, and residues around it similarly determine orientation

Question 4

ER export

Answer 4

Sec12 localised on ER membrane, and is a Sar1-GEF

Sec23/24 dimers are co-incidence detectors of Sar1-GTP and DxD diacidic motifs

Soluble cargo use cargo receptors

Sec13/31 dimers form the structural component, polymerises to complete the COP-II coat, buds off ER exit site

GTPase activity of Sar1 mediates uncoating

Fuses with ERGIC

Question 5

Retrieval of ER proteins

Answer 5

Arf-1 GTPase recruits COP-I coatomer to ERGIC

Soluble ER proteins have C-terminal HDEL/KDEL sequence that is recognised by Erd2, cargo receptor, which recruits COP-I coatomer

HDEL/KDEL is necessary and sufficient for ER retention
(eg. BiP deletion of HDEL, adding HDEL to invertase)

Dibasic motifs of integral TM ER proteins directly recruit COP-I coatomer

COP-I coatomer itself becomes a coincidence detector for Arf-1 GTP and dibasic motifs

Question 6

Sorting of enzymes within Golgi

Answer 6

Kin recognition - but conflicts with cisternal maturation as complexes are too large to enter anterograde complexes

Cholesterol and lipid content - increases from cis to trans, which could affect affinity of the transmembrane domain

Selective retrograde by COP-I vesicles - involves GOLPH3, which interacts with cytoplasmic tails of Golgi enzymes, PI4P, COP-I coat

Question 7

Intra-Golgi transport

Answer 7

Vesicular transport - static compartments, characteristic enzymes. Secretory cargo transported cis-to-trans

Cisternal maturation - dynamic compartments that mature, with characteristic enzymes transported trans-to-cis in COP-I coated vesicles.

Vesicle budding from Golgi initially explained by vesicular transport
Live cell imaging shows maturation.

Question 8

Endosomal Pathway

Answer 8

EE receives cargo from endocytic vesicle, and eventually stop receiving cargo.

Matures to become LE

Acidification by H+ pumps

Formation of intralumenal vesicles, and outward budding to recycle proteins to TGN

Homotypic fusion of LEs

Question 9

LE and lysosome fusion

Answer 9

Kiss-and-run occurs initially

Fusion occurs later to form endolysosome

Lysosomes reformed by maturation, while LE contents are recycled by budding

Question 10

Constitutive uptake of clathrin-mediated endocytosis

Answer 10

Transferrin (Tf) and LDL receptor undergo Constitutive uptake

Transferrin receptor has canonical AP-2 binding motif YXXΦ

LDL receptor is recognised by Dab2, which recruits AP-2.

Upon fusion with EE, acidification mediates cargo unloading.

Receptor is recycled by geometry-based sorting involving recycling endosome

Question 11

Clathrin-mediated endocytosis

Answer 11

AP-2 recruited to cytoplasmic face of receptor either directly or by an alternative adaptor

AP-2 recruits free triskelia and assembles into clathrin lattices

Invagination –> Clathrin-coated pit –> GTp hydrolysis by dynamin mediates pinching off

Uncoating by auxilin and HSC70, which hydrolyzes ATP

Question 12

EGFR endocytosis

Answer 12

Binding of EGF –> ubiquitination of EGFR –> Epsin –> AP-2 –> Clathrin-mediated endocytosis –> Fusion with EE –> ESCRT machinery recognises ubiquitin,

ESCRT0/I/II coral receptors and prevent recycling

ESCRT III polymerisation drives inward budding, forming intralumenal vesicle

Terminates signalling, enables digestion

Question 13

Lysosomal hydrolases

Answer 13

ER targeting, N-linked glycosylation

In cis-Golgi, signal patch mediates modification to form M6P signal

In TGN, M6P signal recognised by M6PR

GGAs or AP-1 adaptor proteins mediate clathrin coating, requiring Arf1

AP-1 recognises same motifs as AP-2

Fuse with early endosomes, pH decreases, dissociation of hydrolase, empty M6P receptors are returned in retromer-coated vesicles

Can also take alternate trafficking route via secretory pathway to PM, then endocytic pathway to EEs

Question 14

Coincidence detection

Answer 14

AP-2 binds PI(4,5)P2 and YXXΦ (or an alternative adaptor)

Budding off clathrin-coated vesicle –> PIP2 is dephosphorylated –> lowers AP-2 binding affinity, aids uncoating

Sec23/24 dimer binds diacidic motifs and Sar1-GTP on ER surface

Question 15

Rab Identity of EE, LE

Answer 15

Rab5-GTP on EE - recruits Rab5-GAP, Rab7-GEF.

Aids maturation of LE, which becomes coated by Rab7-GTP

Question 16

PIP identity for PM and uncoating of clathrin vesicles

Answer 16

Clathrin recruits phosphotase for PI(4,5)P2, so AP-2 decreases binding affinity for the vesicle, aiding uncoating

Question 17

Tethering factors

Answer 17

Long rod-like coiled coil on Golgi and EE.
Tethers from a long distance (eg. EEA1)

Multi-protein complexes - short-distances, promotes fusion and can directly interact with SNAREs

Recognises target vesicles or compartments through vesicle coats, Rab-GTPases, or PIPs

Question 18

Recruitment of tethering machinery to EE

Answer 18

Rab5-GEF on early endosomes activate Rab5-GTP

Recruits effectors Rabaptin5, which stabilises Rab5 and Vps34, a PI3K which increases PI(3)P levels

EEA1 is recruited by PI3P and Rab5

Question 19

SNAREs - function

Answer 19

Minimal machinery for fusion

Activated by MPCs

Confer specificity

Question 20

Dissociation and recycling of SNAREs

Answer 20

a-SNAP binds the trans-SNARE complex

NSF undergoes ATP hydrolysis to break up SNARE complex

SNAREs are returned to original compartment in an inactive state

Question 21

SNARE binding and example

Answer 21

Conserved helical domain - SNARE domain

Interact by forming stable coiled-coil complexes, zipping up to form a 4-helical bundle known as a trans-snare complex

vSNARE synaptobrevin on vesicle binds tSNARE SNAP-25 on PM in neurons