MT3- endo Flashcards
Where is the decision maker point for sorting of proteins in pathways?
TGN- adapter proteins important for this e.g. AP3 for ALP pathway to lysosome, or AP1 or Gga 1/2 (CPY bound to VPS10)
TGN sorting station for?
- Sorting of newly synthesised lysosomal proteins
- Sorting between constitutive and regulated secretory pathways
- Sorting to apical and basolateral membranes
WHat can interfer with TGN sorting?
viruses e.g. HIV
HIV uses endocytic pathway how?
- major histocompatibility complex (MHC) class 1 (antigen presenting to cytotoxic T-cells )- binds more to AP1 so material gets degraded, avoiding immune response.
- Tethrin downregulation- enhances binding to AP1 also. This causes viral retention else so can’t spread.
Mocha mouse mutation? phenotype?
AP3 or AP1- tyrosine oxidase not secreted which is used to make melanin, hence the ligher colour. Also has immune problems
Different motifs that are recognised by endosomal adapter proteins? (3)
Tyrosine based YxxΦ
- lysosomes, basolateral, somatodendritic domains. e.g. Transferin receptors
Dileucine based- e.g. Ach transporter, Kex2,
Ubiquitin
recognition of AP1 proteins by what? Also need?
The mew subunit of AP1 and also needs PI4P.
COPII used for?
ER to Golgi
AP2 used for?
From the PM to early endo.
COPI used for?
Through the golgi.
Retromer used for?
late endosome to golgi e.g. recycling Vps10.
AP4 used for?
TGN to endosome
AP5
late endosome to golgi retrival. Recyling components
Defects in AP-4 or AP-5? Why?
Neurological problems,
e.g. recessive LOF of AP4= spastic paraplegia, fever sensitive seizures, and development delays.
Found my whole genome sequencing.
Poor removal of aggregates in neurons
Gga1 and 2 used for?
late golgi to late endosome, e.g. CPY bound to VPS10
ALS mutation?
in AP4? CHECK
what are MCS?
Inter-membrane contact sites- denser areas where organelles contact. 30nm apart no actual fusion but tethered proteins connect them.
e.g. ER touching PM, endosomes
Who first saw MCSs?
Porter and Palade 1956- protein bridges connect ER and endosome under electron microscope.
Purpose of MCSs? (3)
- Provide a platform for signaling
- Lipid exchange between organelles
- Important for muscle Ca stores- myopathies if defected
What are the two problems with adapter protein sorting?
Proteins need a very specific cellular location but
a. Cargo present in more than one location
b. μ subunits recognizes similar signals e.g. YxxPhi recognised by AP1 and AP2
Doesn’t help specificity.
Solution to the problems of u subunits recognising similar signals etc?
CO-INCIDENCE DETECTION.
not only the adapter but also PI’s.
Location of different Rabs on organelles?
Rab 4- early endosome to PM Rab 5-Pm to Early endo Rab 7- early endo to MVB Rab 11- early endo to recycling compartment Rab 9- MVB to TGN Rab 1,6- Golgi
AP2 structure?
alpha-
Beta subunit- clathrin box to bind clathrin
u- cargo binding motif of YxxΦ or Phi
YxxΦ (or Phi)
is what?
the cargo motif that AP2 u subunit recognises. (and AP1)
Phosphoinositide binding with adapters?
AP1 needs PI4P
AP2 needs PI(4,5)P2
Why is coincidence detection important for enodcytic pathway?
Phosphoinsositides are in low abundance but define organelle identity.
Adapter recognises PI first then sees cargo with the motif.
PI- ensures correct destination- just another quality control step.
Also creates specific zones within the organelle as welll.
AP2 KO?
In yeast- survive- shows there is a bigger picture and suggests redundancy
Evidence that the adapter proteins may not tell the whole story?
- AP2 KO yeast- still survive.
- EGF uptake AP2 KO doesnt affect RNAi.
- The finding of CLASPS
ER contact site regulation example?
Wu et al 2018. ER tubules contact other organelles e.g. surround the areas where cargo buds off an endosome, helping it to bud off.
Evidence of distinct localisation of Adaptors?
Robinson (2004) Immunoflouroscence. Cell fixed, AB specific to complexes, and secondary antibodies. Shows very strong distinct localisation of adapters with little overlap.
How are specific PIs ensured at right location?
Lots of kinases and phosphatases that can convert between phosphoinisitol. The enzymes are key that the right PIs are at the organelle.
Experimentally how is location of PI seen?
Detect an enrichment- follow localisation. Phosphoinositide location can be seen by GPF tagged to the PI binding domains.
AP1 and AP2 and PI’s also bind?
Many other proteins also, especially PI(4,5)P2 involved in co-incidence detection. Role in coated-Pit formation
RNAi KO of AP2
Scotty Robinson-
if give radioactive transferrin over time more and more is uptaked. KD clathrin or AP2 basically stop, showing is necessary for uptake.
Whereas EGF only Clathrin KD reduces uptake.
Theory of alternatives to adapter proteins?
CLASPS act as alternative adapters, bind clathrin, cargo and PI’s. Some also do bind AP2 adapters also.
Example for an alternative adapter than AP2 etc
B-Arrestin alternative adapter essential for GPCR uptake- target for 40% of prescription drugs.
Why have alternative adaptors?
Allows more specificifiy for particular cargo. Can upregulate or down an adapter if lots of one cargo type etc.
PI at PM?
PI(4,5)P2 (think at pm as ccan be broken down to IP3 and DAG)
PI at early endosome?
PI3P (stage 3 up from lysosome, late endo, then early)
PI at TGN?
PI4P(one before early endo- 4th stage back)
PI(3,5)P2 role?
important sorting signal (look up)
What are Rabs? location?
Small GTPases, members of Ras superfamily, distinct subcellular location, recycle between membrane and cytosol.
