Lecture 9 - The endocytic pathway Flashcards
When are the COPI and COPII coats used?
COPI retrograde transport
COPII anterograde transport
What are the four different types of Endocytosis?
- Phagocytosis e.g. macrophages
- cell membrane deforms and engulfs the particle, internalises then the membrane reforms - Pinocytosis e.g. plasma membrane recyling
- surface of the cell extends and loops back on itself, bringing extracellular fluid in - Receptor mediated endocytosis a)signalling e.g. EGF receptor b) nutritional e.g. LDL/transferrrin receptor
- linked to constituative exocytosis
- pulls membrane inside cell - Secretory vesicle endocytosis
Why endocytose?
- Cell needs to maintain cell volume
- Needs to stop signalling (transient acute response to hormones) e.g. adrenal receptor fight/flight - internalise/degrade
What criteria define endocytosis?
- lipid composition e.g. cholersterol, raft components Sphingolipids
- Fission mechanism e.g. Dynamin, CtBP3/BARS, Lipid based
- Regulation e.g. Ubiquitination, Kinases, phosphotates, GEFs, GAPs
- Actin cytoskeleton e.g. Actin, Actin binding proteins, Actin regulatory proteins
- GTPases e.g. CDC42, ARF6, RhoA
- Coat componants e.g. Clatherin, Caveolin, Flotillin, Adapters
What are the clatherin dependent endocytic pathways~?
Constituative
Ligand Induced
These are both Clatherin dependent
What are the clatherin independent pathways?
Dynamin dependent -Caveolar -RhoA-regulated Dynamin independent -CDC42 regulated -ARF6 regulated
What are the principles of Endocytosis?
- Deform the membrane
- Include or Exclude proteins/lipids in the ‘budding’ compartment
- ‘Pinch off’ the compartment from the plasma membrane (energetically unfavourable)
- Uncoat the vesicle so it can tether SNARE and fuse to where it is targeted and deliver the cargo to another compartmen
What proteins are involved in membrane deformation?
- Amphipathic helices (insert proteins into the membrane to cause a slight membrane deformation)
- Loop insertions (pushes lipids apart relative to the bottom forming a slight curve)
- Classic lattices
- BAR domain proteins (Proteins with a BAR domain e.g. FCHO proteins, bind to Pi- and pull membrane up)
What are the mechanisms of clatherin dependent endocytosis?
1-clatherin (heavy and light chains) lattice forms around vesicle, collection of hexagons and pentagons
2-bind to phospholipids of plasma membrane (PtdIns(4,5)P2)
3-nucleation and early, middle, late stages of invagination
4-fission through binding of dynamin (with GTPase activity) to ‘pinch off’ the endosome
5-Uncoating
What are the functions of Epsins?
Epsins (ENTH domain with amphipathic helix)
- involved in Cargo selection
- amphipathic helices can ‘wedge’ can insert into membrane to cause deformation
Give an example of membrane deformation
- 1-BAR domain proteins e.g. FCHO1,2 nucleate at the plasma membrane and allow slight deformation of the plasma membrane
- Epsins (ENTH domain with amphipathic helix), allows cargo selection, amphipathic helix can ‘wedge’ into the membrane to cause further deformation
- Coat assembly helps the membrane to deform furhter,the binding of Amphiphysin encircles the base of the forming bud
- Dynamin binds and pulls the endosome apart from the plasma membrane
- Uncoating by synaptojanin
How are proteins involved in retrograde transport?
Don’t have targeting signals to direct them anywhere but the plasma membrane post golgi, but do have signals to bring them back in
What motifs allow proteins to bind to an adapter protein (AP2) for endocytosis?
On C- terminal tails of membrane proteins:
- YXXΦ motif (tyrosine, XXX, bulky hydrophobic (Valine, Phenolalanine, isoleucine, methionine, leucine)
- an acidic patch dileucine (asparagine/glutamate),XXX, (leucine/isoleucine)
What are the subunits of AP2?
α, β2, δ2, μ2
- α appendage can bind proteins with BAR domain and their wedges
- clatherin binds proteins that also bind to the α appendage
How is clatherin-dependent endo cytosis acheived?
through sequential protein-protein interactions at the site of vesicle budding
- ligand binds receptor which is bound to the AP complex and a large number of accessory proteins (bind cargo, AP2 and clatherin)
- then bind clatherin
What is the process of AP2 binding?
- Ap2 in ‘locked’ conformation (sites for binding not available in closed conformation (δ2/α, μ2) docks to the plasma membrane
- AP2 transition to the open conformation through binding 2 molecules of (PtdIns(4,5)P2) and consequently YXXΦ or an acidic patch dileucine on ‘cargo’ via the now ubobstructed binding pockets on the δ2/α, μ2 subunits
What is the process of Clatherin mediated endocytosis?
- Extended interlog interactions allow clatherin triskeleion to self-assemble into regular latticies
- Adapter protein complexes (AP2) can simaeltaenously bind clatherin, membrane lipids and membrane proteins
- prodcues clatherin coat
What protein is involved in the process of ‘pinching off?’
Dynamin
How is dynemin involved in pinching off?
- hexameric GTPase
- forms a ring arounf the neck of the budding vesicle, pulling it taught, eventually resulting in fission
- highly GTP dependent
- co-ordinates other endocytic proteins through proline rich domain
How can the action of dynamin be shown experimentally?
If attach tubules onto membranes so that they are fixed at either ends in a relaxed manner
- addition of dynamin ‘twists up’ the membrane by moving against itself
- and by continued twisting created ‘knots’ which when enough tension is reached leads to fission
What are the two potential fates for internalised proteins after fusing with early endosome, and what are their purposes?
- Maturation to late endosome (near Golgi, more acidic than early endosome and different Rabs to EE, then fusing with trafficking to lysosome for degradation
- for switching off of a signal to regulate hormone signals e.g. insulin - Recycled to plasma membrane via transport vesicles that bud from the EE and can fuse with recycling endosomes e.g. transferring or the LDL receptor
What is an initial vesicle
vesicle formed from any form of endocytosis and goes on to fuse with an early endosome
pH6.6
What is an early endosome
-vesicle derived from endocytosis
-fuses and delivers contents to late endosome
- defined based upon contents: small GTPase - Rab5
pH5.5-6.
What is a late endosome defined by
small GTPase Rab 7
pH5.5
