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Cell Biology > Lecture 9 - The endocytic pathway > Flashcards

Lecture 9 - The endocytic pathway Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

When are the COPI and COPII coats used?

A

COPI retrograde transport

COPII anterograde transport

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2
Q

What are the four different types of Endocytosis?

A
  1. Phagocytosis e.g. macrophages
    - cell membrane deforms and engulfs the particle, internalises then the membrane reforms
  2. Pinocytosis e.g. plasma membrane recyling
    - surface of the cell extends and loops back on itself, bringing extracellular fluid in
  3. Receptor mediated endocytosis a)signalling e.g. EGF receptor b) nutritional e.g. LDL/transferrrin receptor
    - linked to constituative exocytosis
    - pulls membrane inside cell
  4. Secretory vesicle endocytosis
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3
Q

Why endocytose?

A
  • Cell needs to maintain cell volume
  • Needs to stop signalling (transient acute response to hormones) e.g. adrenal receptor fight/flight - internalise/degrade
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4
Q

What criteria define endocytosis?

A
  • 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
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5
Q

What are the clatherin dependent endocytic pathways~?

A

Constituative
Ligand Induced

These are both Clatherin dependent

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6
Q

What are the clatherin independent pathways?

A 
Dynamin dependent
-Caveolar
-RhoA-regulated
Dynamin independent
-CDC42 regulated 
-ARF6 regulated
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7
Q

What are the principles of Endocytosis?

A
  1. Deform the membrane
  2. Include or Exclude proteins/lipids in the ‘budding’ compartment
  3. ‘Pinch off’ the compartment from the plasma membrane (energetically unfavourable)
  4. Uncoat the vesicle so it can tether SNARE and fuse to where it is targeted and deliver the cargo to another compartmen
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8
Q

What proteins are involved in membrane deformation?

A
  • 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)
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9
Q

What are the mechanisms of clatherin dependent endocytosis?

A

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

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10
Q

What are the functions of Epsins?

A

Epsins (ENTH domain with amphipathic helix)

  • involved in Cargo selection
  • amphipathic helices can ‘wedge’ can insert into membrane to cause deformation
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11
Q

Give an example of membrane deformation

A
  1. 1-BAR domain proteins e.g. FCHO1,2 nucleate at the plasma membrane and allow slight deformation of the plasma membrane
  2. Epsins (ENTH domain with amphipathic helix), allows cargo selection, amphipathic helix can ‘wedge’ into the membrane to cause further deformation
  3. Coat assembly helps the membrane to deform furhter,the binding of Amphiphysin encircles the base of the forming bud
  4. Dynamin binds and pulls the endosome apart from the plasma membrane
  5. Uncoating by synaptojanin
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12
Q

How are proteins involved in retrograde transport?

A

Don’t have targeting signals to direct them anywhere but the plasma membrane post golgi, but do have signals to bring them back in

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13
Q

What motifs allow proteins to bind to an adapter protein (AP2) for endocytosis?

A

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)
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14
Q

What are the subunits of AP2?

A

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
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15
Q

How is clatherin-dependent endo cytosis acheived?

A

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
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16
Q

What is the process of AP2 binding?

A
  1. Ap2 in ‘locked’ conformation (sites for binding not available in closed conformation (δ2/α, μ2) docks to the plasma membrane
  2. 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
17
Q

What is the process of Clatherin mediated endocytosis?

A
  • 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
18
Q

What protein is involved in the process of ‘pinching off?’

A

Dynamin

19
Q

How is dynemin involved in pinching off?

A
  • 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
20
Q

How can the action of dynamin be shown experimentally?

A

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
21
Q

What are the two potential fates for internalised proteins after fusing with early endosome, and what are their purposes?

A
  1. 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
  2. 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
22
Q

What is an initial vesicle

A

vesicle formed from any form of endocytosis and goes on to fuse with an early endosome
pH6.6

23
Q

What is an early endosome

A

-vesicle derived from endocytosis
-fuses and delivers contents to late endosome
- defined based upon contents: small GTPase - Rab5
pH5.5-6.

24
Q

What is a late endosome defined by

A

small GTPase Rab 7

pH5.5

25
Q

What is the lysosome defined by

A

lamp1 (lysosome associated membrane protein 1)
VATPase - pumps protons into lysosome
pH 5

26
Q

What is the process of transferrin recycling to the plasma membrane

A
  1. Transferin binds to iron
  2. Transferrin receptor in membranes binds to transferrin bound to iron, all is internalised through tetrapeptide internalisation sequence binding (YXXΦ) to AP2
  3. low pH causes dissosiation of transferrin from iron
  4. Iron is then redistributed inside the cell
  5. Transferrin and transferrin receptor get recyled out to plasma membrane
  6. once in plasma membrane transferrin dissosiated as the receptor affinity is higher when iron is bound
27
Q

What is the process of LDL receptor recycling to the plasma membrane

A
  1. LDL receptor is translated in the ER and transported out to the plasma membrane where it is presented on the surface
  2. LDL particles bind to LDL receptor via ApoB protein and is endocytised through a clatherin dependent mechanism
  3. pH through the endosomes decreases and dissociates LDL particle from its receptor; the inital vesicle pH6.6, endosome has a pH5.5, lysosome pH4.5-5.
  4. Particle is degraded and the cholesterol is transferred to the ER and inserted into membranes and LDL receptor is taken back out to the cell surface to bind more LDL particles
28
Q

How is the recycling of LDL receptors and endocytosis of cholesterol regulated?

A

Through feedback mechanism
-an increase in the amount of cholesterol leads to a decrease in the level of LDL receptors and enzymes involved in their synthesis

29
Q

Give an example of an inherited LDL recepot trafficking disorder

A

Familial hypercholesterolemia (high serum cholesterol)
-LDL receptors are present on the membrane but do not assosiate with clatherin coated pits, other receptors are trafficked normally
-hypercholesterol in blood
Due to one of two defect:
-mutation of NPXY motif
-mutation of AP2 protein

30
Q

What is the process of EGF-R receptor mediated endocytosis

A
  1. EGF binds to EGF-R and associates with clatherin coated pits through AP2 binding to YXXΦ motif
  2. receptor and ligand are internalised (also relies upon the tyrosine kinase activity of the receptor, also initiating signalling)
  3. transported through EE, LE and lysosome where it is degraded
  4. this leads to receptor downregulation
31
Q

What percentage of unoccupied EGF receptors are found on the plasma membrane/endosome?

A

plasma membrane - 70-18%
endosomes - 20-30%

reversed for occupied receptors

32
Q

How can you experimentally distinguish between recycling and degredation?

A
  1. Chill cells to 4 degrees C and add radiolabelled ligand e.g. transferrin (recycled) or EGF (degraded)
    2.Cells warmed to 37 degrees C and as they are warmed measure the amount of ligand having come into the cell = amount of endocytosis
    -amount of ligand coming up increases then decreases for both
    -if look inside cell cannot tel, add precipitant to the media outside the cell (e.g. tCA) precipitates whole proteins not amino acids
    EGF = no precipitate amino acids remain -> meaure radioactivity
    Transferrin = precipiate measure radio activity

Cell Biology (15 decks)

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