Hettema lecture 5

Question 1

Describe the Pulse-chase experiment by George Palade in 1967.

Answer 1

• undertaken on pancreatic tissue
• tissue was labelled with leucine (3H - radioactive)
• incubated for specific times
• overlaid with emulsion and stored in dark for 3-4 weeks
• processed
  RESULTS:
• ER - 3mins (pulse)
• Golgi - 17mins (chase)
• Plasma membrane - 37min (chase)

Question 2

Is It at a higher/lower temp that temperature sensitive mutants in the ER fold correctly?

Answer 2

Lower temps around 32 degrees (can then move to Golgi etc)

Question 3

How can the arrival of certain proteins in the cis-Golgi from the ER be studies?

Answer 3

Glycosylation studies
- m.w of proteins in Golgi are different than those in ER because they have lower m.w.’s because they have lost a mannose sugar during modification

Question 4

Give the 4 steps in vesicular transport.

Answer 4

1) accumulation of cargo at vesicle point and coat assembly
2) budding
3) uncoating
4) docking and fusion w/ target membrane

Question 5

Name the three different types of coat proteins known.

Answer 5

• Clathrin
• COPI
• COPII

Question 6

Coat assembly is under the control of a ___ase

Answer 6

GTPase

Question 7

Vesicle formation at ER:
Sar1___ bind S__12 on ER
___ exchange for GTP anchors ___ onto the ER membrane

Answer 7

Sar1 GDP binds Sec12 on ER

GDP exchange for GTP anchors Sar1 onto the ER membrane

Question 8

Describe what happens after the docking of SarIGTP to the ER membrane

Answer 8

SarIGTP drives the polymerisation of soluble coat factors leading to the budding of the vesicle. Sorting signals in cargo receptors are recognised by the coat proteins.

Question 9

What process initiates uncoating?

Answer 9

GTP hydrolysis

Question 10

Which 2 SNAREs are involved in creating a tight complex between the vesicle and target membrane?

Answer 10

vSNAREs and tSNAREs

Question 11

How does the vesicle fuse with the plasma membrane?

Answer 11

Complex formed between Rab.GTP and the Rab effector (n target membrane)
SNARE complex between v and tSNAREs

Question 12

How does the SNARE complex disassemble?

Answer 12

ATP hydrolysis of NSF and alpha-SNAP causes the dissociation of the SNARE complex. The SNARE complex is then recycled back to vesicles via endoscopes

Question 13

Why do NSF and alpha-SNAP mutants accumulate vesicles as they act after fusion?

Answer 13

the SNARE complex cannot be broken down if there is no ATP hydrolysis therefore vesicle remains attached to membrane