Adaptor proteins & ENDOCYTOSIS 1

Question 1

The TRANS GOLGI NETWORK (TGN) – why is it needed? Where are the places proteins may need to be sorted to?

Answer 1

The TRANS-GOLGI network is needed to sort newly synthesised proteins to the correct destination. Exmples include:

• Between the regulated and constituative secretion pathways
• Between the apical and basolateral membranes

Question 2

ADAPTOR PROTEINS - why are they needed and why are they specific?
Some examples

Answer 2

Adaptor proteins are needed to sort different cargo at the TGN. Lots of different coats for vesicles are seen at different compartments of the TGN. Different Adaptor proteins are specific to different compartments and particular cargos.
EXAMPLES INCLUDE - AP1, AP3 and GGA

Question 3

HIV1 in protein sorting - - how does the HIV1 virus interfere with TGN sorting?
WHat happens to TETHERIN protein?

Answer 3

Some viruses interfere with TGN sorting

• The HIV1 virus interferes with presentation of MHC (major histocompatibility complex) which is needed to activate T cells, a very important aspect of the immune system.
• Causes them to be targeted to the lysosome and degraded
• Also prevents TETHERIN from binding which is a protein that ‘spits out’ viruses from the cell

Question 4

THE MOCHA MOUSE - what is it and why is it useful?

Answer 4

Mouse with natural AP1 mutations that cause change a change in the mouse coat colour and immune system problems.
The mouse has due to the mutations a deficiency in tyrosine hydroxylase which produces melanin (hence change in coat colour)

Allows us to understand how AP1 and adaptors in general work

Question 5

AP1 / GGA adaptors.

What motifs do they recognise and what are these motifs involved in (examples of motifs)?

Answer 5

These adaptors recognise specific motifs for specific targeting. These motifs include

• TYROSINE BASED motifs involved in recognition at lysosomes, basolateral, somatodendritic domains and transport of APP
• DILEUCINE BASED - found in endo-lysosmal transmembrane proteins, Specialised basolateral endosomal compartments
• For AP1, signals recognised by µ subunits

Question 6

AP4/5 - what can defects in these proteins cause?

Answer 6

AP4 mutants = neurological problems
Mutations cause premature termination of AP4 production which results in seizures and neuronal problems

AP5 mutations cause deficiency in sorting of cargo at the TGN which can result in progressive spastic paraplegia.

Question 7

What are inter membrane contact sites? (MCSs) – what is their function and how do they come about?

What can deficiencies in these cause?

Answer 7

Electron dense areas where organelles touch. The ER contacts many organelles within the cell which is why these are needed.

They form where protein components of the membrane are extended, allowing bridges to form. This provides a platform for SIGNALLING and non-vesicular lipid exchange.

Question 8

2 main endocytic pathways and what are they needed for?

What is this helped by

Answer 8

Clathrin-dependant and clathrin-independant endocytosis.
Clathrin dependant involves AP2 adaptor proteins
Needed for delivery of cargo to the early and then late endosome
» HELPED BY PH GRADIENT

Question 9

What occurs in clathrin-mediated endocytosis?

Answer 9

AP2 recognises µ motif allowing clathrin to bind to adaptor protein. These adaptor complexes have precise sub cellular localisation

Question 10

Adaptor proteins allow precise sub cellular localisation so that specific cargo can be sorted to a specific destination.
A problem is that cargo is often present in more than one location and µ subunits recognise similar signals - If it can bind to more than one set of cargo that will be all round the cell.
How is this problem overcome?

Answer 10

By use of Phosphoinositide binding – recruits protein to the right cargo!

Question 11

What does an adaptor protein recognise and why?

What name is given for this?

Answer 11

• A signal
• Phosphoinositide
  By recognising both of these determinants this ensures the correct adaptor is associated with a protein for correct sorting through the trans golgi network.
  This is called CO INCIDENCE DETECTION

Question 12

Why are lipid kinases and phosphatases in the cell important ?

Answer 12

• Targets proteins to the correct place
• Fine tunes how material taken into a cell
• Ensures membranes are enriched in the right proteins

Question 13

PI4P and PI4,5P

Answer 13

AP1 - binds PI4P

AP2 - binds PI4,5P2

Question 14

How can we visualise PI4P binding domains and what does this allow us to do?

Answer 14

Couple PI4P to GFP

Allows us to see distribution of PIs in the cell

Question 15

Several lines of evidence suggested that AP2 is not solely responsible for cargo recruitment

AP2 and cargo recruitment – what was shown in RNAi knockdown studies?

Answer 15

AP2 (-/-), cargo uptake was still seen – AP2 not needed for that particular cargo?

Not solely AP2 responsible for cargo recruitment

Question 16

Several lines of evidence suggested that AP2 is not solely responsible for cargo recruitment

What was shown in Yeast AP2 (-/-) models? What does this suggest?

Answer 16

No cargo recruitment defect was seen

Not solely AP2 responsible for cargo recruitment

Question 17

CLASPS - what are they and what are their function?

Answer 17

CARGO SPECIFIC (clathrin associated) PROTEINS 
Link clathrin (in clathrin-dependant endocytosis) to the membrane and PIs/cargo/AP2
Behave as alternative adaptor proteins >> perform similar sorting and assembly functions at the clathrin bud site

Question 18

Alternative adaptors - what are they needed for?

Answer 18

• Needed in regulation of uptake of cargo (in endocytosis)
• Allow specificity of uptake

IMPORTANT IN PHYSIOLOGY AND DISEASE