Vesicle Trafficking Flashcards
How can you visualise the endocytotic pathway?
Protein tracking via GFP
What is GFP?
A B-barel protein with 11 B strands and a central alpha helix which absorbs blue light and emits green light.
What is the defect in a class A mutant, and how does this present?
Transport into the ER –> accumulation in the cytosol
What is the defect in a class B mutant and how does it present?
Budding of vesicles from the rER –> Accumulation in the rER
What is the defect in a class C mutant and how does it present?
Fusion of transport vesicles with the Golgi –> accumulation in the ER to Golgi transport vesicles
What is the defect in a class D mutant and how does it present?
Transport from Golgi to secretory vesicles –> accumulation in the golgi
What is the defect in a class E mutant and how does it present?
Transport from secretory vesicles to the cell surface –> accumulation in secretory vesicles
What is the average size of a COP vesicle?
60-80nm
How is Arf1 activated?
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.
What is the COPII sorting signal?
Tyrosine coupled to a di-acidic domain
Outline KDEL receptor
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
Outline SNARE targeting and docking
Priming - exposition of SNARE and tether factors
Tethering - via tether factors
Formation of trans-SNARE complexes
Membrane fusion
What are the 5 mediated protein transport pathways from the TGN?
COP1 retrieval pathway AP3 coated vesicles Clathrin/AP1 coated vesicles Consitutive secretion Regulated vesicle secretion
Outline clathrin/AP1 attachment to membrane
Arf-GDP converted to Arf-GTP. Associated with the membrane. AP1 binds to Arf-GTP and MPR. Clathrin binds to AP1
How is the M6P signal created?
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.
Where does the M6P signal send a protein to?
The lysosome
What are the types of endocytosis?
Macropinocytosis
Clathrin coat
Pinocytosis
Outline receptor mediated endocytosis.
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.
Outline LDL uptake
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.
What is the sorting signal for LDL receptors?
NPXY
How is LDL receptor pH dependent?
The surface of the Beta propellor domain becomes positively charged at pH 5 and then binds to the ligand binding arm
Outline the transferrin cycle
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.
