endocytosis

Question 1

what is the role of clathrin coated vesicles?

Answer 1

responsible for transport of material from the plasma membrane and between the endosomal and Golgi compartments

Question 2

what are the general characteristics of clathrin mediated endocytosis?

Answer 2

• occurs in nearly all cells
• very dynamic
• CCVs are the main route for receptor-mediated endocytosis

Question 3

what is the mechanism for formation of clathrin coated vesicles?

Answer 3

• coat subunits are made of clathrin - they assemble on the surface of the plasma membrane to form the coated ‘plasma bud’
• as more clathrin coats assemble, it causes further distortion of the membrane
• CCVs bud off the plasma membrane
• coat disassembly uncoats vesicles, which fuse with the early endosome

Question 4

how do phosphoinositides regulate vesicle formation?

Answer 4

• inositol phospholipids undergo rapid cycles of phosphorylation and dephospho rylation to produce PIPs
• the localisation of PIPs is highly compartmentalised, and the distribution of these often defines specialised membrane domains

Question 5

what is the structure of a clathrin coated vesicle?

Answer 5

a CCV is formed from 36 triskelia

each triskelia consists of 3 clathrin heavy chains and 3 clathrin light chains

Question 6

what is the position of adapter proteins in the CCV?

Answer 6

they form a discrete inner layer of the coat, positioned between the clathrin cage and membrane

Question 7

what is the function of adapter proteins in the CCV?

Answer 7

they bind the clathrin coat to the membrane and trap transmembrane proteins that bind soluble cargo

Question 8

give an example of a receptor that is recruited into a clathrin coated pit

Answer 8

LDL receptor binding LDL

Question 9

what causes familial hypercholesteraemia?

Answer 9

mutations to the tyrosine present on the ligand binding arm of LDL receptor. instead, there is a cysteine residue here. the ectodomain of LDL receptor binds to LDL, but the receptor does not cluster into clathrin coarted pits

Question 10

what finding shows the importance of clathrin for protein transport?

Answer 10

clathrin can be visualised in the trans-golgi by fluorescence microscoppy

Question 11

what is AP1?

Answer 11

the adapter protein that links cargo to the Golgi

Question 12

how is dynamin involved in pinching off of vesicles?

Answer 12

dynamin contains a PI(4,5)-P2 binding domain which tethers the protein to the membrane
GTP hydrolysis via the GTPase binding domain causes constriction around the neck of the vesicle, allowing it to be pinched off

Question 13

how can you demonstrate that GTP hydrolysis is important for dynamin function and vesicular budding?

Answer 13

• visualise cells incubated with non-hydrolysable GTP by immunogold labelling
• when GTP hydrolysis cannot occur, the long vesicle neck forms but vesicles cannot bud off

Question 14

what is required for uncoating of CCVs?

Answer 14

ATP hydrolysis mediated by Hsp70 chaperone and co-chaperone auxilin

Question 15

what are the properties of the early endosome?

Answer 15

• tubular organisation
• located mostly in the peripheral cytoplasm
• pH of 6

Question 16

why is the pH of the early endosome important?

Answer 16

affects the rate at which ligands move through the endocytic pathway
-acid pH separates LDL from its receptor

Question 17

what is the role of transferrin? what is apotransferrin?

Answer 17

transferrin carries iron in the blood and is called apotransferrin when in the unbound form

Question 18

how does the fate of EGFR differ based on the strength of the signal?

Answer 18

• when levels of EGF are low, EGFR is incorporated into clathrin coated vesicles, these vesicles uncoat, and EGF is recycled to the cell surface
• when levels of EGF are high, this pathway is diverted and EGFR is internalised in a pathway independent of clathrin

Question 19

what is the effect of ubiquitination for EGFR?

Answer 19

targets it for destruction in the lysosome