vesicular trafficking from ER -> Golgi -> ER

Question 1

ER exit site (ERES)

Answer 1

ER subdomain involved in the formation of transport vesicles destined for the Golgi
- usually located next to the cis Golgi
- proper packaging of vesicles with the correct cargo destined for the golgi

Question 2

COP proteins

Answer 2

• a layer of soluble coat proteins attached to the outer surface of the transport vesicles
  2 main functions…
  1. recognize and concentrate specific components to be incorporated into the budding vesicle
  2. mediate ERES membrane curvature and formation of the budding vesicle

Question 3

3 major classes of coat proteins

Answer 3

COP II: moves anterograde direction, from ERES to Golgi
COP I: moves retrograde direction, from Golgi to ERES OR backwards within the golgi
Clathrin: moves anterograde and retrograde, from Golgi OR PM to endosomes

Question 4

what is Sar1 protein

Answer 4

• A G-protein recruited to the cytosolic surface of the ERES responsible for COP II vesicle formation

Question 5

how does Sar1 mediate COP II transport vesicle formation

Answer 5

• Sar1-GDP is recruited to the ERES and binds to Sec 12 (a GEF) which generates Sar1-GTP (active)
• Sar1-GTP gets integrated into the outer leaflet of the ERES which causes the ERES membrane to curve outwards
• Sar1 recruits Sec23 and 24 to the ERES membrane to promote further curvature, followed by Sec13 and 31 to act as the outer scaffold coat for the vesicle

Question 6

Vesicle targeting and fusion at the CGN

Answer 6

1. recognition of the incoming vesicle at the recipient membrane is mediated by Rab proteins. Rabs associate with the membrane in a GTP dependent manner
2. docking of the vesicle at target membranes is mediated by SNARE proteins. specific v-SNAREs interact with t-SNAREs to form a SNARE complex
3. fusion of the vesicle and target membranes, allows for release of vesicle proteins into lumen of acceptor
4. disassembly of SNARE complexes

Question 7

what are Rab proteins

Answer 7

• responsible for recognition of incoming vesicles and recipient membranes in vesicle transport to CGN
• RAB-GTP gets incorporated into vesicles and when the vesicle fuses at CGN membrane RAB-GTP is incorporated into it

Question 8

what are SNARE proteins

Answer 8

• responsible for docking of vesicles at target membranes (especially at CGN)
• cytosolic facing domains are involved in SNARE-SNARE protein binding
• together Rabs and SNAREs contribute to specificity of vesicle targeting
• SNARE complex pulls vesicle and recipient membranes closer together

Question 9

2 classes of SNARE proteins

Answer 9

v-SNAREs: located on transport vesicles
t-SNAREs: located on acceptor membranes

Question 10

What is the fate of vesicle-specific proteins or proteins that “escape from the ER?

Answer 10

• returned to the ER from the CGN by specific retrieval signals
• transported back in COP II vesicles

Question 11

what is the purpose of retrograde transport from CGN to ER

Answer 11

• serves to recycle membrane and return v-SNAREs and Rab proteins that serve in COP II vesicle transport
• uses v and t-SNAREs, as well as Rab and Rab effectors to prevent fusion of COP II vesicles with the EER membrane

Question 12

process of ER protein retrieval involving a KDEL sequence

Answer 12

• resident soluble ER proteins possess a C-terminal KDEL sequence (retrieval sequence)
• escaped ER proteins in the CGN are recognized by the KDEL receptor (in the CGN)
• the luminal facing domain of the KDEL receptor binds to the KDEL sequence of the escaped protein
• the cytosolic-facing domain of the KDEL receptor binds to COP I components
• COP I coated vesicles return the ER protein-KDEL receptor complex back to the ER
• at the ER, KDEL receptor releases the resident ER protein and returns to the CGN

Question 13

what role does pH play when returning ER resident proteins from the CGN

Answer 13

in the CGN, higher [H+] which allows protein with KDEL sequence to bind the KDEL receptor
in the RER, lower [H+] which allows the KDEL receptor to release the ER-resident protein

Question 14

retrograde transport and KKxx

Answer 14

used for the transport of escaped ER membrane proteins
- most resident ER membrane proteins have a cytoplasmic facing C-terminal dilysine (KKxx) sequence
- KKxx serves as an ER retrieval sorting signal
- proteins that cycle between the ER and CGN contain the KKxx retrieval signal
- KKxx is recognized by COP I at the CGN, COP I coated vesicles bud off the CGN and dock/fuse with the ER

Question 15

structure of the golgi complex

Answer 15

• contains membrane-bound cisternae
• CGN - cis cisternae - medial cisternae - trans cisternae - TGN

Question 16

Golgi matrix

Answer 16

mediates the organization of the Golgi complex (stack)
- consists of golgi peripheral and integral membrane proteins
- gold matrix proteins link Golgi to the cytoskeleton

Question 17

GRASPs

Answer 17

• serve as tethering proteins to link different golgi sub-compartments together
• depletion of GRASP complex results in disassembly of the golgi complex

Question 18

GMAP-210

Answer 18

facilitates ER to cis-Golgi communication via a direct interaction with the microtubules between these 2 organelles

Question 19

Golgins

Answer 19

• long filamentous proteins that tether parts if the golgi to the cytoskeleton and other sub cellular compartments
• dimerize and are regulated by G-proteins to anchor them to the Golgi membrane
• if not expressed, the golgi would lose communication with the cytoskeleton and other sub cellular compartments for transport and communication

Question 20

what can cause the golgi to become disordered

Answer 20

• lack of solid interaction with the cytoskeleton (lack of golgins and GRASPs)

Question 21

Cis golgi network

Answer 21

• the destination for material from the ER
• has interconnected tubules and vesicles adjacent to the ERES
• site of COP I vesicle assembly for retrograde transport to the ER
• site of anterograde transport from CGN to the rest of the golgi
• destination of COP I vesicles from cis cisternae

Question 22

Golgi cisternae

Answer 22

• 3 or more large, flattened cisternae divided into cis, medial and trans
• location of golgi metabolism

Question 23

Trans golgi network

Answer 23

• serves as a sorting station: involved in anterograde transport of materials from golgi to other compartments
• site of clathrin vesicle assembly for transport to endosome or lysosome
• site of secretory vesicle assembly for transport to the plasma membrane
• site of COP I vesicle assembly for transport back to trans cisternae

Question 24

functions of the golgi complex

Answer 24

• processing plant of the cell
• synthesis of complex polysaccharides
• modification of proteins and lipids (e.g. glycosylation)
• transport and sorting of proteins
• sorting of proteins typically occurs at CGN or TGN

Question 25

Glycosylation at the Golgi

Answer 25

• N-linked and O-linked
• cis, medial and trans golgi cisternae possess unique glycosyltransferase enzymes which sequentially modify the oligosaccharide core
  N-linked: synthesis begins in ER, modified in golgi
  O-linked: synthesis/modification entirely in Golgi

Question 26

Modifications to target proteins in Golgi to the lysosome

Answer 26

Step 1
- in the cis Golgi cisternae, mannose on the core oligosaccharide is phosphorylated.
- GlcNAc phosphotransferase recognizes a signal patch in the protein meant to be targeted to the lysosome
- N-acetyl glucosamine-1-phosphate is transferred to a specific mannose residue
Step 2
- in the medial Golgi, N-acetlyglucosamine group is removed by phosphodiesterase
- the lysosomal enzyme now contains mannose-6-phosphate (identifying feature!)
- M6P is recognized by M6P receptors in the TGN and the enzyme is sorted to lysosomes

Question 27

pH-dependent delivery of lysosomal proteins

Answer 27

• M6P receptor/lysosomal enzyme is incorporated into clathrin-coated lysosomal vesicles and targeted to lysosome
• pH > 6 in the Golgi which allows lysosomal protein to bind M6P receptor
• pH < 6 in the lysosome which causes the protein to be released from the M6P receptor