MVU11 - INTRACELLULAR TRAFFICKING - 2

Question 1

why are the two mechanisms that ensure that vesicles transport their contents to the correct acceptor membrane?

Answer 1

• Rab GTPase proteins which provide specificity of vesicle targeting and attachment to acceptor membrane
• SNARE fusion proteins provide specificity during fusion of vesicles with acceptor membrane

Question 2

how is the GA organised?

Answer 2

into a stack of membranes
cis (closest to ER)
medial
trans (closer to PM)

Question 3

what is the old model of the transport through the GA?

Answer 3

vesicles transport cargo between layers

Question 4

what is the new model of transport through GA?

Answer 4

each layer matures and becomes the next layer
trans makes the vesicles that go out, runs out of membrane, disappears
medial becomes the new trans one
cis is made by the new incoming vesicles

Question 5

where do CCV carry cargo to?

Answer 5

to PM and endosomes

Question 6

what maintains the organisation of the stack?

Answer 6

a cytosolic protein matrix

Question 7

how are N-linked glycans modified in the GA?

Answer 7

removal of mannoses and addition of different sugars, often with negative charges
other complex sugars are attached to Ser and Thr side chains: O-linked glycosylation
many different combinations: specificity, heterogeneity

Question 8

what is the effect of glycosylation in the GA?

Answer 8

promotes protein folding:
makes folding intermediates more soluble (prevents aggregation)
sequential modifications -> progression of folding or degradation
sugars have limited flexibility ->protect from proteases, stabilise protein structure (protective coat in the cytosol)
signaling hubs (regulations of development)
rich in OH groups, can be phosphorylated

Question 9

what is the function of proprotein convertases?

Answer 9

some PM and extracellular proteins are made as a longer inactive form in the ER
cut by proprotein convertases into a shorter active form at the GA
proteases recognise specific patterns of AAs (not specific motifs)
cleavage activates protein by removing inhibitory region

Question 10

what is an example of a protein made active by being cut short?

Answer 10

proinsulin made into an active polypeptide
convertases remove the middle section, the two remaining sections form the active insulin
prevents premature signaling by insulin at the ER
disulfide bonds bring together C and N termini

Question 11

what is an example of a protein in the signalling pathway that is activated by being cut?

Answer 11

ATF6 is activated by proteolysis in the GA
regulation is by trafficking
BiP covers ER exit signal on ATF6
proteases are only present in the GA

Question 12

what are Rab proteins?

Answer 12

large sub-family of Ras-related proteins
different organelles and vesicle types in secretory pathway have unique sets of rab proteins

Question 13

what does Rab-GTP bind to?

Answer 13

binds to a large number of Rab-effector proteins which mediate vesicle targeting

Question 14

what are the different functions of Rab?

Answer 14

1. rabs can act at several steps in vesicle targeting
2. assist cargo selection and coat formation during vesicle budding
3. connect vesicle to motors on cytoskeleton for transport
4. tether vesicles to acceptor membrane –> specificity
5. recruit SNARE fusion protein

Question 15

what are the specific rabs at each vesicle transport stage of the secretory pathway?

Answer 15

Rab1: ER to GA
Rab 2: GA to ER
Rab7,9: GA and early to late endosome
Rab5: endocytosis from PM to enosome
Rab4,11: recycling from endosome to PM
Rab3: exocytosis of secretory vesicles

Question 16

explain the Rab membrane anchor

Answer 16

Rabs have two prenyl lipid groups attached at their C termini
in the GDP bound state Rab is soluble and not associated with the membrane -> lipid is covered up by other proteins (GDI, GDF)
in the GTP bound state lipid modifications are exposed and anchor Rab to the membrane
the Rab effector proteins become attached to membrane through Rab-GTP

Question 17

how is Rab activated?

Answer 17

specific GEF on membrane produces anchored, active Rab-GTP
GEF is linked to formation of vesicle coat

Question 18

how does RabGTP work through vesicle proteins?

Answer 18

attach vesicle to motor proteins
tether vesicle to target membrane
activate PI kinases and GEFs to make more Rab GTP in clusters on acceptor membrane

Question 19

what is the vesicle Rab cycle?

Answer 19

1. Vesicle Rab is activated by
   GEF on donor membrane
   and packaged onto vesicles
   – some interact with cargo,
   assist uncoating, or attach
   to motors
2. Vesicle Rab-GTP binds
   specifically to tethers on
   acceptor membrane
3. After fusion,GAPs on target
   membrane inactivate Rab
4. Inactive vesicle Rab-GDP is
   recycled through cytosol to
   donor membrane

Question 20

what runs through the cytosol and provides structure to the cell?

Answer 20

protein filaments (actin) and microtubules (tubulin)
connected to each other and anchored to organelles and PM (cortical cytoskeleton)

Question 21

what are characteristics of actin filaments?

Answer 21

shorter, clustered at PM, highly crosslinked into network

Question 22

what are characteristics of microtubules?

Answer 22

longer, thicker, organised around centrosome near nucleus

Question 23

how do motor proteins transport vesicles?

Answer 23

along cytoskeleton
myosins on actin, dyneins and kinesins on microtubules
different families and mechanisms but ATP dependent
(dyneins are AAA proteins, other motors are not)

Question 24

what do motors do?

Answer 24

bring vesicles to general location of acceptor membrane but do not provide targeting specificity

Question 25

what are vesicle tethers?

Answer 25

Rab effectors: long proteins which connect vesicle with acceptor
tethers are bound to specific vesicle on Rab on one end and specific membrane on the other end
first determinants of vesicle targeting specificity

Question 26

what are the types of tethers and where do they act?

Answer 26

tethers of different structural families act at different organelles
coiled coil tethers: mostly within GA and on endosomes
multisubunit tethers: act on other compartments
ER to/from GA
GA to PM
endosome and lysosome

Question 27

what is the structure of coiled coil tethers and what do they do?

Answer 27

• Dimers with coiled-coil structures (pairs
  of α-helices) that remain assembled
  after use
• Coiled-coil tethers form the Golgi matrix:
  – maintain the organization of the Golgi
  stack
  – connect vesicles to Golgi
  – anchored to membrane, or
  attached by GTPases
  – long filaments with multiple Rab
  binding sites

Question 28

what are multisubunit tethers? and what are the different families for different organelles?

Answer 28

Most secretory pathway steps
involve multisubunit tethers
* Different structural families:
– ER to Golgi, within Golgi
(TRAPPI, II)
– endosome and lysosome
(HOPS/CORVET)
– ER and PM: CATCHR family
(exocyst, GARP, COG, Dsl1)

Question 29

how does the TRAPPI tether work?

Answer 29

ER to GA
1. TRAPPI acts as GEF for Rab1 on Golgi
2. Rab1 binds coiled-coil tether
3. CC tether binds to vesicle and hands vesicle to TRAPPI, closer to
membrane
4. TRAPPI helps organize SNAREs

Question 30

how does the CATCHR family work?

Answer 30

exocyst at PM
1. Before tethering but after uncoating, some subunits are on either
vesicle or PM
2. Complete 8-mer complex forms tether
3. May bring SNARE to membrane to start fusion

Question 31

how do endosome tethers work?

Answer 31

To endosomes: Rab5 and CORVET
* To lysosomes: Rab7 and HOPS
* Complexes have the same core subunits
* Different end subunits bind different Rabs
* Also bind SNARES