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BMS376 Membrane trafficking > Endocytosis > Flashcards

Endocytosis Flashcards

1
Q

Parts of the Golgi?

A
  1. cis
  2. med
  3. trans
  4. TGN- decisionmade at this point either go to cell surface or lysosomes
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2
Q

What are adaptor proteins important for?

A

Sorting cargo.

the more cargo the more proteins

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3
Q

What do AP1 and AP3 and GGAsdecide?

A

What’s performing and where cargo is going to from TGN

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4
Q

TGN sorting station what does it do?

A
  1. Sorts newly synthesised lysosomal proteins
  2. sorting between constitutive and regulated secretory pathways
  3. sorting of apical and basolateral membranes
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5
Q

What and how do viruses affect the TGN?

A

Interfere with sorting- become diverted

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6
Q

HIV leads to downregulation of 2 things?

A
  1. MHC class 1- important for immune system, has sorting which allows more binding of AP1 which leads to lysosomes (avoid mounting immune response)
  2. Tetherin- enhances binding of AO AP1 to be degraded
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7
Q

Difference between normal mice and mocho mice? WHY?

A

Mocho= light coat colour, naturally occurring mutation in AP1 and AP3
- deficiency in tyrosine hydroxylase= less melanin

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8
Q

Motifs that are recognised by AP1 and GGAs?

A
  1. tyrosine based:
    - 1/xx phi
    - AP1 signals recognised by U subunit
    - requirement for PI4P
  2. Dileucine based:
    - xxxL - endo-lysosomal transmembrane, specialized basolateral
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9
Q

What is a motif?

A

a distinctive sequence on a protein or DNA, 3D structure that allows binding interactions to occur

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10
Q

How can you define coats?

A

By adaptor proteins

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11
Q

Different types of adaptor proteins and where are they?

A 
AP1- TGN
AP2- PM
AP3- tubular endosome
AP4- TGN
COP1- Golgi stack 
CGA- TGN 
AP5-late endosome
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12
Q

What neurological problems do defects in AP4 and AP5 give rise to?

A

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

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13
Q

What are inner membrane contact sites (mcs)

A

Identified- 1957

  • important for ca2+ release from ER
  • improved by fluorescent tags make ER
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14
Q

What is a contact site?

A

Where 2 membranes come closely together

ER is shown to be in close proximity- close connections

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15
Q

What did Emily eden show?

A
  • protein bridges between the er and endosome

- protein membranes tethered with long cytosolic domain bridges

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16
Q

What are the functions of mcs?

A

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

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17
Q

fundamental mechanism of membrane cytosis? (2 types)

A
  1. clathrin dependent- always see clathrin coated pits, see structures, do biochemistry
  2. clathrin independent- controls uptake
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18
Q

Why do they have 2 different pathways for uptake?

A

If one doesn’t work then you use the other and still survive

- redundancy or specificity

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19
Q

What is the pathway for clathrin dependent or independent uptake

A
  1. deliver to EE

2. dissociation of signalling and ligands, sorting- recycle or degrade

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20
Q

What is PH important for/

A

Lysosomes

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21
Q

How does clathrin mediated endocytosis work?

A

coats forms for structural rigidity as bud forms

Clathrin doesn’t bud directly to membrane, needs an adaptor (AP2)

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22
Q

Sorting signals in endocytic proteins

A
  1. tyrosine based YxxPhi- TFR
  2. Dileucine based XXXL- acetylcholine transporter
  3. FXNPXY- LDL receptor
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23
Q

What do adaptor proteins show?

A

A precise subcellular localisation

AP1 and AP3 overlap location- shown with immunofluorescence

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24
Q

Problem with maintaining subcellular localisation

A

a) cargo present in more than one location

b) u subunits recognise similar signals

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25
Q

Solution to maintaining subcellular localisation

A 
Phosphoinositide binding 
- define organelle identity 
- low abundance phospholipid 
AP1= PI4P
AP2= PI(45)P
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26
Q

What is coincidence detection?

A

recruiting components to right destination in cell

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27
Q

Example of coincidence detection

A

AP1 goes to TGN, primary determinant is 6C PI4P is there, protein has yxxo motif and gets bound
Allows pit to form

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28
Q

What do Rabs and PIs help to define?

A

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

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29
Q

What are the lines of investigation that show that AP2 is not solely responsible for cargo recruitment?

A
  1. KO of all AP2 subunits
  2. RNAi studies in mammalian cells
  3. characteristic of CLASPs
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30
Q

Experiment where coated vesicles are made and ligand measure ligand uptake in control and siRNA treated cells
RESULTS

A

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
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31
Q

What do these results show?

A

They show that for EGF ligand AP2 is not the key adaptor molecule for the cargo

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32
Q

What do clathrin adaptors bind?

A

Clathrin, cargo and phosphoinositides

some also bing AP2

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33
Q

Where are PI3P and PI4P subcellular binding domains?

A 
PI3P= early endosomes important for sorting 
pP14P= tagged with GFP, see clusters near nucleus
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34
Q

What is the Rab family?

A
  • 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
35
Q

What is the Rab cycle?

A

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

36
Q

What is the rab GDP/GTP cycle?

A

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

37
Q

What is GDI?

A

Chaperone- scoop Rab in GDP form out of membrane

38
Q

What is Rab effector?

A

Tether, recognises incoming vesicles, get SNARE complex fusion , Rab switched off and recycle

39
Q

What is GAP?

A

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

40
Q

localisation and function of Rab5?

A

localises to endocytic pathway

role in endosomal fusion

41
Q

What can help to define function of Rab5?

A

Mutant forms - Rab5GDP or RabGTP

- define domain on endocytic pathway through localised areas enriched in Rab5

42
Q

What is used to segregate domains in the endosomal compartment?

A

PI and transferrin uptake

compare low density lipoproteins

43
Q

What happens when Rab5Q79L is overexpressed?

A

Forms large endosomes

44
Q

Tethering proteins can often be…

A

Rab effectors

45
Q

Rab5 regulates multiple stages on events at the EE pathway

A

a) sorting
b) uncoating
c) motility
d) tethering
e) fusion

46
Q

Rab 5 in the tethering step

A

Rab5-GDP- Rab5-GEF which joins PI3K and covalently attaches lipid by the rab effector to the rab5 membrane domain

47
Q

What happens in an experiment where GST-Rab5 pull down with cytoplasmic enzyme GST-X?

A
  • 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
48
Q

Molecules shown to be Rab effectors?

A
  • 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)
49
Q

How does Rab5 select out proteins?

A

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
50
Q

How do we get first Rab5?

A

GEF driver for initial recruitment

51
Q

How big is the first domain?

A

Stop getting bigger by recruiting GAP

52
Q

What is super resolution microscopy? What does it show?

A

Look below the defraction of limit of life

Shows- EEA1 and internalised EGFR

53
Q

How is cargo delivered to the endosomal compartment?

A

Bud out of cytoplasm to lumen
pinch off forming vesicle
MvB- limiting membrane and lots of vesicles inside

54
Q

How is cargo then moved to late endosome?

A
  • ubiquitin is a signal- degradation

- fusion with ubiquitin inhibits recycling of TFR and the receptor interacts with HRs

55
Q

What is ubiquitin?

A

Signal can be for degradation

reversible post translational modification

56
Q

How can you show ubiquitin is a signal?

A

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

57
Q

How does late endosomes containing MVB affect their function?

A

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)
58
Q

What machinery allows visualisation of inward binding?

A
  • Electron microscopy of Late endosome shows vesicles budding inwards, dense coat of one microdomain (Rab contribute to this)
  • Topography- reconstruct sections, see MVB
59
Q

What does transport from EE to LE involve?

A

ESCRT- composed of HRs and STAN

HRs= adaptor as recognises cargo that has ubiquitin attached and bins PI3P, recruit to endosomes

60
Q

How is clathrin involved in transport from EE to LE?

A

flat clathrin lattice important for efficient sorting of HRs for cargo incorporated proteins

61
Q

Experiment- used PtdINs(3)P probe redirected from EEA1 positive region into HRs- containing microdomains through clathrin binding- What does this show?

A

EEA1 binds PI3P and Rab5
HRS binds PI3P NOT Rab5
so the 2 are segregated as one binds Rab one doesn’t

62
Q

What are the standard conditions for this PtdIN(3)P experiment?

A

Delete c terminus of HRS so it cant bind clathrin so they can bind in same region as EEA1

63
Q

What are the 4 cytosolic protein complexes?

A

ESCRT 0= binds ubq cargo
ESCRT 1 = recognises
ESCRT 2 = oligomerization of protein
ESCRT 3= cargo concentration and deubiqitination

64
Q

4 cyctosolic proteins transport cargo by?

A

Passing it along
Invagination
sission

65
Q

What are DUBs protein and what problems can occur?

A

Take ubiquitin off protein, helps with recycling in cells

Can get problems in ERADD-misfolding proteins out of ER

66
Q

What do VPS4 ATPase do?

A

Help with budding

viruses hijack and recruit formal vesicles from cell and allow to escape

67
Q

What do ESCRT protein give rise to?

A

mutations cause cancer and neurodegenerative diseases

overexpression and deletion of protein in malignancy

68
Q

Which pathway is activated in membrane trafficking depends on?

A
  1. location
  2. context
  3. signal
69
Q

How does a cell know how to respond?

A
  • regulation of trafficking

- once arrived at MVB- receptor tail no longer accessible outside for signalling so it has been turned off

70
Q

What is signal attenuation in endocytosis?

A
  • removal from cell surface

- signal degradation

71
Q

When does signalling occur?

A

continually signalling

only when incorporated into the MVB does the signal stop

72
Q

How to quantify signal?

A

look at response of rat liver cells to EGF and insulin
EGF- signal occurs in intracellular compartments not PM
Insulin- cell surface response

73
Q

What is TGF?

A

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)
74
Q

What is SMAD2?

A

SMAD2- TF that translocates to the nucleus

- requires endosome associated protein SARA

75
Q

How is signalling determined by route of entry?

A

TGFb are present in caveole and ccps

Paper looks if entry affects TGFb

  • R enter through clathrin= OK
  • R enter through calveole = Degradation
76
Q

What happens when you treat with nystatin?

A

Cholesterol clepleleng enzyme, disrupt association of EEA1 and Receptor

77
Q

What are the affects downstream of using nystain?

A

doesn’t affect clathrin or signalling whereas KCL inhibits clathrin (overexpress mutant dynamin)

78
Q

Explaining dynamin cycle

A

-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

79
Q

How is EGFR sensitive to ligand concentration

A

High- CIE (leads to degradation)

Low- CME (results in signalling)

80
Q

What is the signal that switches the pathway undertaken?

A

Receptor Ubiquitination

81
Q

Different ligands influence receptor trafficking

A

both EGFa and EGF bind EGFR

  • EGFa - promote recycling
  • EGF- promote degradation
82
Q

What is fate of EGF determined by?

A

Rab machinery

  • Rab7 phosphorylation= degradation
  • RCP, Rab11 = recycling
83
Q

What are the 2 possibilities for receptors moving through the endocytic pathway?

A
  • R signal to same effector throughout the pathway

- signalling outputs are qualitively different depending on where they are located

84
Q

Can signalling occur from endosomes under physiological conditions?

A

Yes mutant forms of Rab5 and dynamin test this

known so target EGF

BMS376 Membrane trafficking (5 decks)

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