Lecture 10

Question 1

Why doesn’t the cell want unfolded proteins to leave the ER lumen?

Answer 1

Unfolded proteins tend to form insoluble aggregates that block important cellular functions

Question 2

What is the significance of unfolded proteins retaining a single glucose?

Answer 2

Single glucose has an affinity for calnexin

Question 3

What is calnexin? Function?

Answer 3

Glucose molecule that recognizes glucose molecules on unfolded enzymes and prevents them from being packaged in the Golgi

Question 4

Describe the role of calnexin in ER protein folding.

Answer 4

1. Calnexin binds to glucose of unfolded proteins in ER lumen
   * *prevents proteins fro leaving ER in a vesicle
   * *allows more time for the protein to fold properly
2. Protein folds properly
3. Glucose is removed
4. Calnexin lets go of protein –> allows it to enter vesicles destined for Golgi

Question 5

What is the effect of carbohydrate (sugar) modification on an SDS PAGE?

Answer 5

Higher band in presence of microsomes

Question 6

What is the effect of tunicamycin on an SDS PAGE?

Answer 6

Inhibits glycosylation –> fails to add sugar modification to protein –> protein becomes smaller

Question 7

What is glycosylation?

Answer 7

Sugar modification

Question 8

What is the effect of Triton X-100 on an SDS PAGE?

Answer 8

Causes membrane to be permeable –> no band when protease is present

Question 9

What is the effect of protease on an SDS page? W/ vs. w/o microsomes

Answer 9

Degrades protein.

• w/o microsomes –> no band
• w/ microsomes –> band @ same height as w/o protease

Question 10

GTP-γ-S is a non-hydrolyzable form of GTP. When a G-protein binds GTP-γ-S, it is locked in the GTP conformation. Why is the band in the last lane much smaller?

Answer 10

Ribosome is unable to restart translation at the ER

Question 11

What is the default pathway for proteins in the ER?

Answer 11

Secreted or plasma membrane

Question 12

What are the 3 steps to vesicle transport?

Answer 12

1. Budding
2. Docking
3. Fusion

Question 13

What is the role of coat proteins in vesicle transport?

Answer 13

• Coat proteins form a cage around the cytoplasmic face of a budding vesicle –> provide a scaffold to add curvature to the vesicle membrane to bud it off its donor membrane
• Need to be completely removed before vesicle can dock and fuse w/ its target membrane

Question 14

List the 3 major types of coat proteins.

Answer 14

• COP II coated vesicles
• COP I coated vesicles
• Clathrin coated vesicles

Question 15

Cis vs. Trans Golgi?

Answer 15

• cis = close to ER

- trans = close to plasma membrane

Question 16

In what direction do COP II coated vesicles travel? To and from what organelles?

Answer 16

Anterograde:

-ER to cis-golgi

Question 17

In what direction do COP I coated vesicles travel? To and from what organelles?

Answer 17

Retrograde:

• cis-Golgi to ER
• trans-Golgi to cis-Golgi

Question 18

In what direction do Clathrin coated vesicles travel? To and from what organelles?

Answer 18

• Anterograde: trans-Golgi network to endosomes

- Retrograde: endocytosi (plasma membrane to endosomes)

Question 19

Explain the mechanism of COP II-coated vesicle formation.

Answer 19

1. Sec 12 on ER membrane acts as GEF for Sar1
2. GTP-bound Sar 1 inserts onto ER membrane –> recruits 4 more proteins to form the COP II coat
3. Formation of COP II coat serves as a timer for Sar1 –> 30 sec after coat forms, Sec 23 (from coat) acts as GAP for Sar1
4. GDP-bound Sar1 no longer binds to ER membrane –> coat disassembles

Question 20

Arf1 is the G protein for COP I vesicles that acts analogously to Sar1 for COP II vesicles. Where would you expect to find the GEF for Arf1?

Answer 20

Cis-Golgi membrane

Question 21

What are the 2 classes of proteins that ensure the specificity of vesicle trafficking?

Answer 21

• SNARE

- Rab

Question 22

Where are v-SNAREs found? t-SNAREs?

Answer 22

• v-SNARES: on the membrane of vesicles

- t-SNARES: on the target membranes