Vesicle Trafficking

Question 1

How can you visualise the endocytotic pathway?

Answer 1

Protein tracking via GFP

Question 2

What is GFP?

Answer 2

A B-barel protein with 11 B strands and a central alpha helix which absorbs blue light and emits green light.

Question 3

What is the defect in a class A mutant, and how does this present?

Answer 3

Transport into the ER –> accumulation in the cytosol

Question 4

What is the defect in a class B mutant and how does it present?

Answer 4

Budding of vesicles from the rER –> Accumulation in the rER

Question 5

What is the defect in a class C mutant and how does it present?

Answer 5

Fusion of transport vesicles with the Golgi –> accumulation in the ER to Golgi transport vesicles

Question 6

What is the defect in a class D mutant and how does it present?

Answer 6

Transport from Golgi to secretory vesicles –> accumulation in the golgi

Question 7

What is the defect in a class E mutant and how does it present?

Answer 7

Transport from secretory vesicles to the cell surface –> accumulation in secretory vesicles

Question 8

What is the average size of a COP vesicle?

Answer 8

60-80nm

Question 9

How is Arf1 activated?

Answer 9

It is brought to the membrane and interacts with a GEF. This causes exchange of GDP for GTP leading to extension of the protective arm into the membrane. When it encounters a GAP it is hydrolysed and dissociates from the membrane.

Question 10

What is the COPII sorting signal?

Answer 10

Tyrosine coupled to a di-acidic domain

Question 11

Outline KDEL receptor

Answer 11

Random incorporation of ER luminal proteins with the KDEL signal into COPII vesicles.
KDEL binds to the KDEL receptor in the cis-Golig at low pH
Retrograde transport in COPI vesicles
Release of cargo in the ER at a higher pH

Question 12

Outline SNARE targeting and docking

Answer 12

Priming - exposition of SNARE and tether factors
Tethering - via tether factors
Formation of trans-SNARE complexes
Membrane fusion

Question 13

What are the 5 mediated protein transport pathways from the TGN?

Answer 13

COP1 retrieval pathway
AP3 coated vesicles
Clathrin/AP1 coated vesicles
Consitutive secretion
Regulated vesicle secretion

Question 14

Outline clathrin/AP1 attachment to membrane

Answer 14

Arf-GDP converted to Arf-GTP. Associated with the membrane. AP1 binds to Arf-GTP and MPR. Clathrin binds to AP1

Question 15

How is the M6P signal created?

Answer 15

Glucose N-acetylglucosamine phosphotransferase binds UDP-GlcNAc in it’s catalytic site and a lysosomal enzyme in it’s recognition site. UMP is removed binding GLcNAc and P to the lysosomal enzyme. It is released from GlcNAc phosphotransferase. A phosphodiesterase removes the final group.

Question 16

Where does the M6P signal send a protein to?

Answer 16

The lysosome

Question 17

What are the types of endocytosis?

Answer 17

Macropinocytosis
Clathrin coat
Pinocytosis

Question 18

Outline receptor mediated endocytosis.

Answer 18

AP2 is recrutied to the membrane along with clathrin. A coat assembles and the membrane curves. A coated pit forms. Amphiphysin and Dynamin bind to form a collar which pinches off the vesicle. This detaches and then sheds the coar.

Question 19

Outline LDL uptake

Answer 19

A coated pit forms around the LDL receptors. LDL binds to the LDL receptor. The CCV is pinched off. It sheds it’s coat. The receptors are delivered to the early endosome. In the late endosome there is a pH shift to 5.0 so that LDL dissociates from the receptor. The receptor is recycled back to the plasma membrane. The LDL is sent to the lysosome where it is degraded to cholesterol, fatty acids and amino acids.

Question 20

What is the sorting signal for LDL receptors?

Answer 20

NPXY

Question 21

How is LDL receptor pH dependent?

Answer 21

The surface of the Beta propellor domain becomes positively charged at pH 5 and then binds to the ligand binding arm

Question 22

Outline the transferrin cycle

Answer 22

Ferrotransferrin binds to the transferrin receptor. A clathrin coated pit forms. The vesicle is pinched off and sheds it’s coat. In the late endosome the low pH cause Fe£+ to be released from apotransferrin. Apotransferrin is returned to the pm and released.