Endocytosis Flashcards
Parts of the Golgi?
- cis
- med
- trans
- TGN- decisionmade at this point either go to cell surface or lysosomes
What are adaptor proteins important for?
Sorting cargo.
the more cargo the more proteins
What do AP1 and AP3 and GGAsdecide?
What’s performing and where cargo is going to from TGN
TGN sorting station what does it do?
- Sorts newly synthesised lysosomal proteins
- sorting between constitutive and regulated secretory pathways
- sorting of apical and basolateral membranes
What and how do viruses affect the TGN?
Interfere with sorting- become diverted
HIV leads to downregulation of 2 things?
- MHC class 1- important for immune system, has sorting which allows more binding of AP1 which leads to lysosomes (avoid mounting immune response)
- Tetherin- enhances binding of AO AP1 to be degraded
Difference between normal mice and mocho mice? WHY?
Mocho= light coat colour, naturally occurring mutation in AP1 and AP3
- deficiency in tyrosine hydroxylase= less melanin
Motifs that are recognised by AP1 and GGAs?
- tyrosine based:
- 1/xx phi
- AP1 signals recognised by U subunit
- requirement for PI4P - Dileucine based:
- xxxL - endo-lysosomal transmembrane, specialized basolateral
What is a motif?
a distinctive sequence on a protein or DNA, 3D structure that allows binding interactions to occur
How can you define coats?
By adaptor proteins
Different types of adaptor proteins and where are they?
AP1- TGN AP2- PM AP3- tubular endosome AP4- TGN COP1- Golgi stack CGA- TGN AP5-late endosome
What neurological problems do defects in AP4 and AP5 give rise to?
AP4- recessive loss of function, seizure and epilepsy
- defect is removed of aggregate at end of neuron
-ALS also have mutations in AP4
AP5- spastic parapledgia
What are inner membrane contact sites (mcs)
Identified- 1957
- important for ca2+ release from ER
- improved by fluorescent tags make ER
What is a contact site?
Where 2 membranes come closely together
ER is shown to be in close proximity- close connections
What did Emily eden show?
- protein bridges between the er and endosome
- protein membranes tethered with long cytosolic domain bridges
What are the functions of mcs?
Membrane platform for:
- signalling
- non-vesicular lipid exchange
- mobilisation of calcium stores- defects in ca= defects in muscles
ER tissues promoted for breaking of mitochondria
fundamental mechanism of membrane cytosis? (2 types)
- clathrin dependent- always see clathrin coated pits, see structures, do biochemistry
- clathrin independent- controls uptake
Why do they have 2 different pathways for uptake?
If one doesn’t work then you use the other and still survive
- redundancy or specificity
What is the pathway for clathrin dependent or independent uptake
- deliver to EE
2. dissociation of signalling and ligands, sorting- recycle or degrade
What is PH important for/
Lysosomes
How does clathrin mediated endocytosis work?
coats forms for structural rigidity as bud forms
Clathrin doesn’t bud directly to membrane, needs an adaptor (AP2)
Sorting signals in endocytic proteins
- tyrosine based YxxPhi- TFR
- Dileucine based XXXL- acetylcholine transporter
- FXNPXY- LDL receptor
What do adaptor proteins show?
A precise subcellular localisation
AP1 and AP3 overlap location- shown with immunofluorescence
Problem with maintaining subcellular localisation
a) cargo present in more than one location
b) u subunits recognise similar signals
Solution to maintaining subcellular localisation
Phosphoinositide binding - define organelle identity - low abundance phospholipid AP1= PI4P AP2= PI(45)P
What is coincidence detection?
recruiting components to right destination in cell
Example of coincidence detection
AP1 goes to TGN, primary determinant is 6C PI4P is there, protein has yxxo motif and gets bound
Allows pit to form
What do Rabs and PIs help to define?
Endocytic intermediates
PI
- low abundant pi recruit right protein to right place in cell
- allows visualisation of subcellular distribution eg. yellow fluorescence shows where PIP2 binds
What are the lines of investigation that show that AP2 is not solely responsible for cargo recruitment?
- KO of all AP2 subunits
- RNAi studies in mammalian cells
- characteristic of CLASPs
Experiment where coated vesicles are made and ligand measure ligand uptake in control and siRNA treated cells
RESULTS
Clathrin and AP2 kd
- Sharp uptake of cpm is abolished with both KD when looking at transferrin ligand
- EDF ligand- sharp uptake in AP2 but clathrin KD abolishes uptake
What do these results show?
They show that for EGF ligand AP2 is not the key adaptor molecule for the cargo
What do clathrin adaptors bind?
Clathrin, cargo and phosphoinositides
some also bing AP2
Where are PI3P and PI4P subcellular binding domains?
PI3P= early endosomes important for sorting pP14P= tagged with GFP, see clusters near nucleus
What is the Rab family?
- member of Ras superfamily
- have distinct subcellular location
- cycle between membrane and cytosol
- required for fusion
- approx. 60 in humans
- most ubiquitinately expressed- lots of proteins
What is the Rab cycle?
Rab GTPase and v snare of PM - forms transport vesicle- rab effector helps docking by forming snare complex with V and T snares- membrane fusion - GTP hydrolysis with soluable rab-GDP - guanine exchange factor takes Rab back to pm
What is the rab GDP/GTP cycle?
Rab GTP —- GDP by RabGEF embedded in the membrane
RabGDP—-GTP by RabGAPs
mediate exchange of Rab, vesicle pinch off and go to target membrane
What is GDI?
Chaperone- scoop Rab in GDP form out of membrane
What is Rab effector?
Tether, recognises incoming vesicles, get SNARE complex fusion , Rab switched off and recycle
What is GAP?
GTPase activating protein
- rab has low intrinsic rate of hydrolysis so needs GAP to activate and switch GTP to GDP
Used not constituently active so can happen faster
localisation and function of Rab5?
localises to endocytic pathway
role in endosomal fusion
What can help to define function of Rab5?
Mutant forms - Rab5GDP or RabGTP
- define domain on endocytic pathway through localised areas enriched in Rab5
What is used to segregate domains in the endosomal compartment?
PI and transferrin uptake
compare low density lipoproteins
What happens when Rab5Q79L is overexpressed?
Forms large endosomes
Tethering proteins can often be…
Rab effectors
Rab5 regulates multiple stages on events at the EE pathway
a) sorting
b) uncoating
c) motility
d) tethering
e) fusion
Rab 5 in the tethering step
Rab5-GDP- Rab5-GEF which joins PI3K and covalently attaches lipid by the rab effector to the rab5 membrane domain
What happens in an experiment where GST-Rab5 pull down with cytoplasmic enzyme GST-X?
- look at proteins that specifically bind to X
- GTPyS- conditions where locked in GDP form as they cant hydrolyse allowing only proteins binding to the GTP form to be found
Molecules shown to be Rab effectors?
- Rabaptin= fused with vesicle, role in endosomal dynamics
- EEA1= long coil coil protein that acts as a tethter for incoming endocytic pathway with endosomes
- Rabex 5= GEF bound indirectly through rabaptin (piggy back)
How does Rab5 select out proteins?
Rab establishes membrane microdomains
- Active rab recruit effector with GEF to generate more Rab GTP
- PI3K on gel= defines endosomes
- Pi3k+ Rab5 = effectors that cluster in coming vesicles can recognise and promote fusion
How do we get first Rab5?
GEF driver for initial recruitment
How big is the first domain?
Stop getting bigger by recruiting GAP
What is super resolution microscopy? What does it show?
Look below the defraction of limit of life
Shows- EEA1 and internalised EGFR
How is cargo delivered to the endosomal compartment?
Bud out of cytoplasm to lumen
pinch off forming vesicle
MvB- limiting membrane and lots of vesicles inside
How is cargo then moved to late endosome?
- ubiquitin is a signal- degradation
- fusion with ubiquitin inhibits recycling of TFR and the receptor interacts with HRs
What is ubiquitin?
Signal can be for degradation
reversible post translational modification
How can you show ubiquitin is a signal?
Identify site and mutate it - show sufficiency (cargo determines number of vesicles)
Add protein that wouldn’t normally go to lysosomes and see if it goes there- necessity
How does late endosomes containing MVB affect their function?
cargo recruit proteins to specific place
- sorting in limiting membrane= recycling or lysosomal membrane
- sorting in MVB= degradation (once incorporated into the intraluminal vesicle its targeted for degradation)
What machinery allows visualisation of inward binding?
- Electron microscopy of Late endosome shows vesicles budding inwards, dense coat of one microdomain (Rab contribute to this)
- Topography- reconstruct sections, see MVB
What does transport from EE to LE involve?
ESCRT- composed of HRs and STAN
HRs= adaptor as recognises cargo that has ubiquitin attached and bins PI3P, recruit to endosomes
How is clathrin involved in transport from EE to LE?
flat clathrin lattice important for efficient sorting of HRs for cargo incorporated proteins
Experiment- used PtdINs(3)P probe redirected from EEA1 positive region into HRs- containing microdomains through clathrin binding- What does this show?
EEA1 binds PI3P and Rab5
HRS binds PI3P NOT Rab5
so the 2 are segregated as one binds Rab one doesn’t
What are the standard conditions for this PtdIN(3)P experiment?
Delete c terminus of HRS so it cant bind clathrin so they can bind in same region as EEA1
What are the 4 cytosolic protein complexes?
ESCRT 0= binds ubq cargo
ESCRT 1 = recognises
ESCRT 2 = oligomerization of protein
ESCRT 3= cargo concentration and deubiqitination
4 cyctosolic proteins transport cargo by?
Passing it along
Invagination
sission
What are DUBs protein and what problems can occur?
Take ubiquitin off protein, helps with recycling in cells
Can get problems in ERADD-misfolding proteins out of ER
What do VPS4 ATPase do?
Help with budding
viruses hijack and recruit formal vesicles from cell and allow to escape
What do ESCRT protein give rise to?
mutations cause cancer and neurodegenerative diseases
overexpression and deletion of protein in malignancy
Which pathway is activated in membrane trafficking depends on?
- location
- context
- signal
How does a cell know how to respond?
- regulation of trafficking
- once arrived at MVB- receptor tail no longer accessible outside for signalling so it has been turned off
What is signal attenuation in endocytosis?
- removal from cell surface
- signal degradation
When does signalling occur?
continually signalling
only when incorporated into the MVB does the signal stop
How to quantify signal?
look at response of rat liver cells to EGF and insulin
EGF- signal occurs in intracellular compartments not PM
Insulin- cell surface response
What is TGF?
TGFb- cellular growth and differentiation
- Receptor activation leads to phosphorylated SMAD2
- TGF has to be in endosome to signal
- present in clathrin coated pits and caveole (clathrin independent)
What is SMAD2?
SMAD2- TF that translocates to the nucleus
- requires endosome associated protein SARA
How is signalling determined by route of entry?
TGFb are present in caveole and ccps
Paper looks if entry affects TGFb
- R enter through clathrin= OK
- R enter through calveole = Degradation
What happens when you treat with nystatin?
Cholesterol clepleleng enzyme, disrupt association of EEA1 and Receptor
What are the affects downstream of using nystain?
doesn’t affect clathrin or signalling whereas KCL inhibits clathrin (overexpress mutant dynamin)
Explaining dynamin cycle
-Dynamin, eps15 and kcl inhibit ccp
- Dynamin inhibits caveole
but dynamin, eps15 and KCL activate nystalin
Outcomes
- CCP- EE- signal transduction pathway
- Caveole - caveolin positive vesicle- degradation pathway
This shows you need to be in the endosome to signal
How is EGFR sensitive to ligand concentration
High- CIE (leads to degradation)
Low- CME (results in signalling)
What is the signal that switches the pathway undertaken?
Receptor Ubiquitination
Different ligands influence receptor trafficking
both EGFa and EGF bind EGFR
- EGFa - promote recycling
- EGF- promote degradation
What is fate of EGF determined by?
Rab machinery
- Rab7 phosphorylation= degradation
- RCP, Rab11 = recycling
What are the 2 possibilities for receptors moving through the endocytic pathway?
- R signal to same effector throughout the pathway
- signalling outputs are qualitively different depending on where they are located
Can signalling occur from endosomes under physiological conditions?
Yes mutant forms of Rab5 and dynamin test this
known so target EGF
