Why doesn’t the cell want unfolded proteins to leave the ER lumen?
Unfolded proteins tend to form insoluble aggregates that block important cellular functions
What is the significance of unfolded proteins retaining a single glucose?
Single glucose has an affinity for calnexin
What is calnexin? Function?
Glucose molecule that recognizes glucose molecules on unfolded enzymes and prevents them from being packaged in the Golgi
Describe the role of calnexin in ER protein folding.
- 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 - Protein folds properly
- Glucose is removed
- Calnexin lets go of protein –> allows it to enter vesicles destined for Golgi
What is the effect of carbohydrate (sugar) modification on an SDS PAGE?
Higher band in presence of microsomes
What is the effect of tunicamycin on an SDS PAGE?
Inhibits glycosylation –> fails to add sugar modification to protein –> protein becomes smaller
What is glycosylation?
Sugar modification
What is the effect of Triton X-100 on an SDS PAGE?
Causes membrane to be permeable –> no band when protease is present
What is the effect of protease on an SDS page? W/ vs. w/o microsomes
Degrades protein.
- w/o microsomes –> no band
- w/ microsomes –> band @ same height as w/o protease
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?
Ribosome is unable to restart translation at the ER
What is the default pathway for proteins in the ER?
Secreted or plasma membrane
What are the 3 steps to vesicle transport?
- Budding
- Docking
- Fusion
What is the role of coat proteins in vesicle transport?
- 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
List the 3 major types of coat proteins.
- COP II coated vesicles
- COP I coated vesicles
- Clathrin coated vesicles
Cis vs. Trans Golgi?
- cis = close to ER
- trans = close to plasma membrane
In what direction do COP II coated vesicles travel? To and from what organelles?
Anterograde:
-ER to cis-golgi
In what direction do COP I coated vesicles travel? To and from what organelles?
Retrograde:
- cis-Golgi to ER
- trans-Golgi to cis-Golgi
In what direction do Clathrin coated vesicles travel? To and from what organelles?
- Anterograde: trans-Golgi network to endosomes
- Retrograde: endocytosi (plasma membrane to endosomes)
Explain the mechanism of COP II-coated vesicle formation.
- Sec 12 on ER membrane acts as GEF for Sar1
- GTP-bound Sar 1 inserts onto ER membrane –> recruits 4 more proteins to form the COP II coat
- 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
- GDP-bound Sar1 no longer binds to ER membrane –> coat disassembles
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?
Cis-Golgi membrane
What are the 2 classes of proteins that ensure the specificity of vesicle trafficking?
- SNARE
- Rab
Where are v-SNAREs found? t-SNAREs?
- v-SNARES: on the membrane of vesicles
- t-SNARES: on the target membranes
