Unit 4: ER to Golgi

Question 1

ERES

Answer 1

ER exit sites

• distinct subdomain of the ER
• located next to cis face of Golgi complex

Question 2

ERES is enriched with molecular machinery responsible for ____

Answer 2

• formation of budding of membrane bound vesicles destined for Golgi
• proper packaging of vesicles with the correct lumenal and membrane cargo proteins destined for Golgi

Question 3

Do resident ER proteins enter the Golgi destined transport vesicles?

Answer 3

• prevented from entering Golgi-destined transport

Question 4

What are the three major classes of coat proteins?

Answer 4

1. COPII (anterograde) ER to Golgi
2. COPI (retrograde) Golgi to ER
3. Clathrin (from Golgi or PM to endosomes)

Question 5

Where do COPII-coated vesicle assemble?

Answer 5

ERES

Question 6

Transport Vesicle Assembly at the ERES:

Step One

Answer 6

• soluble COPII component Sar1-GDP (Sar1 = Gprotein) recruited from cytosolic surface of the ERES
• Sar1-GDP binds a guanine-exchange factor, which generates Sar1-GTP

Question 7

What is GEF?

Answer 7

Guanine Exchange factor

- ER integral membrane protein that catalyzes the exhange of GDP for GTP on Sar1

Question 8

Transport Vesicle Assembly at the ERES:

Step Two

Answer 8

• Sar-1-GTP integrates into the ER outer leaflet at ERES

- Results in membrane curvature of the ERES membrane

Question 9

Transport Vesicle Assembly at the ERES:

Step Three

Answer 9

• Sar1-GTP recruits other soluble COPII components
• from cytosol to surface of ERES membrane
• Sec23 and Sec24
  …

Question 10

Transport Vesicle Assembly at the ERES:
Step Three
What happens to Sec 23 and Sec 24

Answer 10

• form a dimer to promote further bending ERES membrane

Question 11

Transport Vesicle Assembly at the ERES:
Step Three
What does Sec24 do?

Answer 11

• Sec24 also binds to cytosolic-facing domains of selected integral membrane including
• membrane cargo proteins
• membrane cargo receptors proteins
• membrane receptor proteins required for trafficking and docking

Question 12

Transport Vesicle Assembly at the ERES:

Step Four

Answer 12

• additional soluble COPII components recruited to cytosol to surface of growing coated vesicle ‘bud’

Question 13

Transport Vesicle Assembly at the ERES:
Step Five
What happens after COPII coat assembly?

Answer 13

• vesicle bud pinches off from ERES and vesicle begins to traffic to proper recipient membrane

Question 14

Transport Vesicle Assembly at the ERES:
Step Five
What happen prior to vesicle fusion with the Golgi?

Answer 14

• COPII coat disassemble
• SarI-GTP is converted back to Sar1- GDP
• and released along with all of the other COPII proteins
• into the cytosol for another round of COPII coat assembly at ERES

Question 15

What is a Rab protein?

Answer 15

• large family of lipid-anchored membrane proteins
• located on all transport vesicles and recipient membranes
• vesicle targeting specificity and unique rabs to different membranes

Question 16

What does the association of the Rab protein with a membrane require?

Answer 16

GTP

Question 17

Vesicle targeting and fusion at CGN

Step 1

Answer 17

Recognition of the incoming vesicle and recipient membranes

Question 18

Vesicle targeting and fusion at CGN

Step 2

Answer 18

Tethering of the incoming vesicle to recipient membrane

Question 19

What do GTP-Rabs do?

Answer 19

• complementary membrane bound
• recruit various recipient factors
• act like tethering proteins
• from cytosol to surface of the vesicle and recipient membranes

Question 20

What are tethering proteins?

Answer 20

• highly elongated (fiber-like, rod-shaped)proteins

- or components of large multi-protein complexes

Question 21

What do tethering proteins do?

Answer 21

• form a molecular bridge

- mediate vesicle membrane-recipient membrane contact by bringing the two membranes close together

Question 22

What is step 3 of vesicle targetting and fusion at CGN?

Answer 22

docking of the vesicle at recipient membranes

Question 23

What mediates the 3rd step of vesicle targetting?

Answer 23

• SNARE proteins help docking

Question 24

What are SNARE proteins?

Answer 24

• large family of integral membrane-bound proteins located on all transport vesicles AND all recipient membranes
• specificity (unique vesicle targeting)

Question 25

What is a SNARE motif?

Answer 25

- all SNARE proteins have a SNARE motif - cytosolic facing domain that extends from membrane surface - SNARE-SNARE protein binding

Question 26

2 main classes of SNARE proteins

Answer 26

v snares | t snares

Question 27

Explain v-SNARES

Answer 27

- vesicle membranes - incorporated into vesicle membrane at site of budding on donor comparment - i.e. at ERES vSNARE incorporated into golgi-destined VESICLE MEMBRANE

Question 28

What are t-SNARES

Answer 28

- target acceptor membranes | - i.e. at the cis Golgi

Question 29

What is the SNARE complex? What mediates it?

Answer 29

v-snare + t=SNARE | - cytosolic facing SNARE motif mediates

Question 30

What is step four of vesicle targeting fusion at CGN?

Answer 30

fusion of vesicle and acceptor membranes

Question 31

What does the SNARE complex do? (4th step of vesicle targeting and fusion)

Answer 31

- pulls opposing vesicle and recipient membranes together = fusion

Question 32

What does the vesicle-recipient membrane fusion result in?

Answer 32

1. Lateral movement of vesicle membrane proteins (cargo and receptors) into recipient membrane 2. Release of vesicle soluble 'cargo' proteins into the interior lumen of the acceptor organelle

Question 33

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

Answer 33

returned from the CGN to the ER by specific ER retrieval signals via retrograde transport

Question 34

What is the direction of retrograde transport?

Answer 34

cis-Golgi to ER

Question 35

What sequence do most resident soluble ER proteins possess?

Answer 35

KDEL sequence

Question 36

What are examples of ER retrieval signals?

Answer 36

``` KDEL sequence KKxx sequence (cytosolic facing) ```

Question 37

What is the role of the KDEL sequence?

Answer 37

recognized in the CGN by KDEL receptor

Question 38

What does the KDEL sequence bind to?

Answer 38

- lumenal side - binds KDEL sequence of escaped protein in CGN lumen - cytosolic side- binds COPI components - releases once reaches ER

Question 39

What is the role of COPI?

Answer 39

- mediates the formation of transport vesicles at the CGN | - return soluble ER protein KDEL receptor complex back to ER

Question 40

What is the role of the KKxx sequence?

Answer 40

- cytosolic facing - sequence on escaped ER membrane proteins at the CGN - recognized by COP1 - COP1 coated vesicles return resident ER to ER

Question 41

What else has a KKxx ER retrieval signal?

Answer 41

KDEL receptor