Organelle Ecology - The Golgi

Question 1

Structure

Answer 1

1. Membrane traffic between compartments
2. ERGIC
3. Cisternal progression

Question 2

Two theories for membrane traffic between compartments

Answer 2

1. the vesicle traffic is shuttling cargo between distinct compartments
2. compartments change identity (mature)

Question 3

Maturation

Answer 3

ER -> Golgi -> PM -> EEs -> LEs

Question 4

What are the dynamics of transport?

Answer 4

• anterograde cargo traffic
• retrograde retrieval
• vesicle addition/loss

Question 5

ERGIC

Answer 5

• ER-Golgi Intermediate Compartment
• vesiculo-tubular cluster
• form via ERES homotypic fusion
• mature as COPI selectively removes escaped ER residents using recycling cargo receptors, vSNAREs

Question 6

vSNARES

Answer 6

important ESPs

Question 7

Imaging VTC formation

Answer 7

1) nocadazole
2) VSVG localises to ERESs
3) nocadazole washout: vesicles migrate -> nuclear region; fuse

Question 8

ERES

Answer 8

ER Exit Site

Question 9

VSVG

Answer 9

vesicular stromatis virus glycoprotein

Question 10

The Golgi

Answer 10

• mobile
• lots!
• connected cisternae
• At: up to 20 compartments!
• flexible identity

Question 11

Cisternal progression?

Answer 11

• early evidence tyasken from large cargoes in coccoliths
• remove scales (new scales formed)
• repeat in a species with small scales (that could feasibly fit into vesicles); scales only present in cisternae

Question 12

coccoliths

Answer 12

• scaled
• too big for vesicles
• <20 cisternae
• each have 1x scale

Question 13

Cisternal progression in animalia is studied in

Answer 13

collagen fibrils

Question 14

collagen fibrils

Answer 14

• ~300nm rods
• too large to fit into vesicles

Question 15

How to study cisternal progression in animalia

Answer 15

1. DPD inhibits proline hydroxylation; ER collagen export
2. DPD washout: collagen fibrils chased cis-trans

Question 16

Classical cisternal progression model

Answer 16

1. ER cargo proteins -(vesicles)-> cis-Golgi
2. coalescence of a new cis-Golgi cisterna
3. dissociation of the trans-most cisterna to PM (as vesicles)

Question 17

Cisternal distinction

Answer 17

• Early/Late
• separate by sucrose density gradient centrifugation
• assay enzymes in each fraction (biochemical)
• quantitative immunogold EM (uneven enzymatic cis-trans distribution/compartmental localisation)

Question 18

mannosidase II

Answer 18

G2-localised

Question 19

secretory cargo

Answer 19

• is consistent throughout the whole of the Golgi
• consistent with cisternal progression

Question 20

Cisternal maturation

Answer 20

• a modification of the cisternal progression model
• cargo remains in cisternae which progress through the Golgi
• COPI retorgrade enzyme recovery (explains distribution)

Question 21

In vitro vesicle transport assays set-up

Answer 21

1) isolate CHO15B mutant membranes
2) add wt Golgi CHO

Question 22

CHO15B

Answer 22

• Chinese Hamster Ovary 15B
• lacks Golgi GlcNAc glycosyltransferase
• infected with VSV
• can only produce VSVGs with immature glycans; sensitive to endo H

Question 23

In vitro vesicle transport assays obs and inference

Answer 23

• VSVG w/ end H-R glycans
• a vesicle intermediate must carry cargo from mutant -> wt compartments for processing

Question 24

What are the assumptions of the vesicular shuttle?

Answer 24

• Golgi cisternae are stable compartments
• anterograde COPI transport

Question 25

Quantitative immune EM

Answer 25

- consistent with cisternal maturation. - .90% of VSVG cargo is cisternal - 10-15x concentration - excluded from vesicles

Question 26

VSVG cargo in COPI vesicles?

Answer 26

- ~5% - may be accidental? - default?

Question 27

COP1 vesicles

Answer 27

- moderately selective (50nm) - particularly for lumenal proteins

Question 28

peri-Golgi COPI vesicles

Answer 28

- KDELr components - ManII

Question 29

cisternal maturation in yeast

Answer 29

- single cisterna

Question 30

Inter-cisternal tubules

Answer 30

- allow small cargoes to move rapidly between cisterna

Question 31

Quantitive immunogold accounting for size

Answer 31

- small: albumen (moves rapidly, ~2min) - large: VSVG trimeric membrane protein (~20min)

Question 32

COPI

Answer 32

- anterograde - ERES -> cis-Golgi

Question 33

Inter-Golgi transport is

Answer 33

mediated by COPI

Question 34

Studying inter-Golgi transport

Answer 34

1) label cells with different Golgi 2) fuse

Question 35

Inter-Golgi transport obs

Answer 35

- small, soluble, membrane-bound, secreted cargo and exogenous Golgi resident enzymes are exchanged via COPI vesicles - large, soluble aggregates traverse individual stacks (no inter-Golgi transport)

Question 36

Pelham's lipid ratchet

Answer 36

- as cisternae mature, sphingolipids are synthesised and accumulate, resulting in membrane thickening - resident proteins with short TMDs partition in earlier cisternae (retrograde) - cargo with longer TMDs partition throughout the stack

Question 37

Golgi-resident enzymes

Answer 37

- responsible for changing membrane lipid type: ER -> Golgi -> PM - increased sphingolipids and cholesterol - cholesterol regulation

Question 38

Multiple mechanisms to sort diverse cargo

Answer 38

1. size 2. cisternal maturation 3. solubility

Question 39

How do the resident Golgi proteins fit into the cisternal maturation model?

Answer 39

- membranes and cargoes mature by successive acquisition of resident Golgi proteins

Question 40

organelle recognition

Answer 40

- lipid (phosphainositide) binding - active GTPases

Question 41

RabGTPase cascade proposes that

Answer 41

vesicles can rapidly change the identity of the organelle from which they have budded

Question 42

RabGTPases

Answer 42

- short-lived (easily controlled @ subcellular level for plasticity and accuracy) - drive sequential changes in organelle identity - important ESPs

Question 43

RabGTPase cascade model

Answer 43

1) RabA GEF: active RabA recruitment 2) RabA recruits RabB GEF etc. - can be intermingled, or in a single compartment

Question 44

GAP proteins

Answer 44

- activated by upstream GTPase - inactivate Rab GTPases - sharpen boundaries between Rab GTPase domains

Question 45

ER identity

Answer 45

Sec12 activates Sar1 GTPase (identity marker)

Question 46

Sec12

Answer 46

resident ER integral protein