L11 - inter-membrane contact sites

Question 1

what are membrane contact sites? (MCS)

Answer 1

close appositions between 2 organelles

Question 2

Are there membrane contact sites between the endoplasmic reticulum and the mitochondria?

Answer 2

• yes

- ER holds the mitochondria like a hand - membrane become very close but do not fuse

Question 3

What does the small lipid anchor have to do with MCS?

Answer 3

• lipid is embedded into the membrane which allows organelles to have affinity/bind to organelles which they are making contact with

Question 4

How can protien tethers (lipid anchors) and bridges be seen between MCS?

Answer 4

• by electon microscopy

Question 5

Give some of the functions of MCS?

Answer 5

-provide a membrane platform for

• signalling
• non-vesicular lipid exchange
• mobilization of calcium stores

Question 6

what organelle is the major calcium store of cells?

Answer 6

ER is major calcium store of cells

- close bringing together of calcium stores via intermembrane contact sites

Question 7

give the major adaptor protein in mammalian cells

Answer 7

AP2

adaptor protein 2

Question 8

Give the fundamental mechanisms of (clathrin dependent) endocytosis

Answer 8

• ligand binds to transmembrane receptor
• ligand-receptor complexes become clustered in clathrin coated pits
• adaptor protein acts as a link between cargo in the vesicle and vesicle coat (clathrin)
• membrane pinched off by dynamin and early endosome forms containing cargo

Question 9

what do rab proteins do?

Answer 9

• key in sorting of the early endosome

Question 10

what rab proteins are abundant on early endosomes?

Answer 10

rab 4

rab5

Question 11

give some clathrin - independent endocytic pathways

Answer 11

• non-coated vesicles
• calveolae
• macropinocytosis
• phagocytosis

Question 12

what are calveolae?

Answer 12

• cup shaped invagination of membranes
• invaginations form rosettes in muscle cells - store
• rosettes are endocytotic - take up cargo

Question 13

what happens in macropinocytosis?

Answer 13

• non-selective form of endocytosis where cells take up the EC environment
• immune cells use this to sample environment they are in

-mobilizaiton of actin forms a pseudopod which can take up EC en. - cells ruffle

Question 14

what does phagocytosis take up?

Answer 14

debris and pathogens

Question 15

what does clathrin dependent endocytosis take up?

Answer 15

• nutrience

- signalling receptors

Question 16

How many subunits do adaptor proteins have?

Answer 16

• 4 subunits

Question 17

which adaptor protein subunit does clathrin bind to?

Answer 17

Beta subunit of adaptor proteins

Question 18

which adaptor proteins bind to clathrin?

Answer 18

AP1 (trans-golgi-network)

AP2 (plasma membane)

Question 19

how many adaptor proteins are there?

Answer 19

AP1-5

Question 20

where is AP5 found?

Answer 20

adaptor protein found on late endosomes (still require sorting)

Question 21

which subunit of adaptor proteins recognises sorting signals?

Answer 21

Mu (μ)

- cargo binding so recognises sorting signals on cargo

Question 22

what is co-incidence detection?

Answer 22

• where adaptor proteins bind to cargo and bind to phosphoinositides
  (both are required which allows co-localisaiton of adaptor proteins)
  -needed as mu subunits recognise similar signals and same cargo/sorting signal may be present in multiple locations.

Question 23

what phosphoinositides do AP1 and AP2 bind to?

Answer 23

• AP1 binds to cargo and PI4P

- AP2 binds cargo and to PI(4,5)P2

Question 24

what helps to define the endocytotic intermediates?

Answer 24

rabs and phosphoinositides

different PIs at different places in the cell- lable with GFP to visualise localisations

Question 25

what are CLASPs?

Answer 25

clathrin associated sorting proteins

Question 26

what are the recognition motifs on Adaptor proteins?

Answer 26

WxxF and DP[FW]

Question 27

what is the dynamin homolgue in drosophila?

Answer 27

shibre
(GTPase - riquired for pinching off of clathrin coated vesicles)
- accumulation of endocytotic intermediates if dynamin cannot function (at high temperatures)

Question 28

what removes the clathrin coat?

Answer 28

hsc70

-clathrin coat needs to be removed to fuse with endosomes