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Cell 600 > Sept 22 - Endocytosis and Lysosomal Targeting > Flashcards

Sept 22 - Endocytosis and Lysosomal Targeting Flashcards

1
Q

What’s a cell free system for studying golgi vesicular transport?

A

Put a donor golgi lacking GlcNAc transferase I (N-acetylglucosamine transferase I) with VSVG. Add this to an acceptor golgi with GlcNAc. You should see viral protein

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2
Q

What is GlcNaC?

A

N-acetylglucosamine transferase I

It comes between Mannosidase I and Mannosidase II in the glycosylation pathway.

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3
Q

What is the step affected by endo h?

A

Mannosidase II

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4
Q

Three ways that sorting can occur

A
  1. Coats
  2. Lipid-dependent partitioning
  3. Geometric - membrane proteins partition into tubes with high SA:V ratio, default recycling for membrane proteins
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5
Q

Rafeldin A

A

Collapses multiple compartments into 1

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6
Q

Acid hydrolases, cathespins, acid proteases

A

Proteins that are found in lysosomes

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7
Q

Re-derived storage (Glut4) compartment

A

Retains glucose transporters in insulin-responsive cells

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8
Q

List some lysosome related organelles

A 
Platelet dense granules
Pigment cell melanosomes
Antigen processing compartments
Mast cell granules
Cytolytic granules in T cells
Secretory lysosomes
Lamellar bodies in lung cells
Weibel-Palade bodies in epithelial cells
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9
Q

What’s the significance of transferrin?

A

You can incubate cells on ice and when you raise to a permissive temperature the cells will endocytose transferrin.
If you label transferrin red transferrin will go to the recycling endosome for 1st 5-10 min and then to sorting endosome for last 5-10 min. This is different from opioid which goes to the late endosome for 1st 5-10 min and then the sorting endosome.

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10
Q

Rab5

A

Enriched on the early endosome

Rab5 GTP recruits Rab7 GEF. Rab7 GTP recruits Rab5 GAP.

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11
Q

Rab7

A

Enriched on the late endosome

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12
Q

Things that Rab5 recruits

A
  • EEA1 - a tethering protein
  • VPS45 - a SNARE regulator
  • Rab7 GEF
  • Motor proteins for positioning on microtubules
  • Lipid kinases (PtdIns 3-kinase)
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13
Q

PtdIns(3)P

A

Rab5 recruits PtdIns 3-kinase to early endosomes. PtdIns 3-kinase is generated. It recruits effectors with PtdIns(3)P binding domains - FYVE domains, PX domains.

Present almost exclusively on early endosomal membranes

3 is lucky, as would be an early end to this lecture…

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14
Q

PtdIns(4,5)P2

A

Enriched in patches at plasma membrane. Recruits or modulates endocytic machinery. Major regulator of actin dynamics.

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15
Q

PtdIns(4)P

A

Enriched at the golgi, required for secretion.

4 is unlucky, Gollum is also unlucky.

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16
Q

PtdIns(3,5)P2

A

Generated on endosomes by PtdIns(3)P 5 kinase - required for late endosomal maturation and autophagy.

3 - endosomal
5 - last number, so late.

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17
Q

What does PtdIns(3)P recruit?

A
  1. Docking proteins for MVB formation
  2. Sorting nexins for tubule formation to allow for endocytic recycling
  3. PtdIns(3)-5 kinase to generate PtdIns(3,5)P2, enriched on late endosomes - this makes sense!
18
Q

How do you get internal membranes?

A
  1. ESCRTs recognize Ub on cargo (I guess it’s been ubiquitinylated)
  2. Cargo is sorted to internal vesicles as they form on the endosomal membrane.
  3. Ub is removed by isopeptidase and is not released into endosome lumen.
19
Q

How is ESCRT-0 recruited to vacuolar domains?

A

By binding to PtdIns(3)P

20
Q

Multivesicular body

A

Process by which ubiquitin tagged proteins enter endosomes (basically the MVBs are multiple vesicles inside one another)
Budding from PM is topologically equivalent to budding into MVB.

21
Q

NPVY

A

Sequence required to internalize LDLR receptor. The tyrosine is essential. Most internalized cell surface receptors have some kind of internalization sequence that is similar.

22
Q

Triskelion

A

Motif consisting of 3 interlocking spirals - 3 heavy chains and 3 light chains. Icosahedral coat.. Formed by clathrin. Honeycomb shape.

23
Q

Traintrack type structures on EM

A

Associated with clathrin

24
Q

Does clathrin interact with the NPxY signal?

A

NO there must be other things in between! `

25
Q

AP-2

A

Adaptor complex. Involved in Endocytosis. Heterotetromeric complex associated with clathrin. This is how Clathrin coated vesicles are able to choose various things to endocytose I think.
4 subunits
They recruit clathrin and bind endocytic and endosomal sorting molecules. So they help to assemble the clathrin coat and gather the phosphatidylinositols.

26
Q

Which protein is AP-2 like at golgi and ER?

A

COPI at golgi and COPIi at ER exit sites.

Cluster of AP-2 recruits clathrin coat, forms pit.

27
Q

What internalization/sorting signals do AP-2 bind to?

A

tyrosine based YxxO - present in the cytosolic domain of many receptors and endosome/lysosome resident integral membrane proteins

di-leucine based (D/E xxx L L/I) - frequently found on lysosomal integral membrane proteins, some internalized proteins

28
Q

If you knock down clathrin and AP-2, what happens to transferrin internalization? What about EGFR or LDLR internalization?

A

KD either one and transferrin internalization drops.
KD clathrin and EGFR and LDLR internalization drops
KD AP-2 doesn’t have any effect on EGFR or LDLR internalizatoin because their internalization/sorting signal must be different

29
Q

What does Shabire mutant tell us?

A

Drosophila dynamin mutant where endocytic vesicles do not break off

30
Q

What is dynamin

A

Large GTP-ase, not in ras superfamily. GTP binding to dynamin closes the ring and constricts the membrane, causing it to pinch off.

31
Q

What do GTPgammaS terminals look like?

A

GTP gamma S treated nerve terminals look like Shabire terminals.

32
Q

Pinchase

A

Something that pops off vesicles

33
Q

GGA

A

Golgi-localized, Gamma ear containing, Arf-binding - not for endocytosis, but for endosomal sorting
Localized to TGN and endosomes - recruited by Arf1.
Recognizes D/E xx L L/I (APs recognize ExxxLL)
Binds clathrin via hinge region.

34
Q

Sorting nexins

A

Characterized by a PX domain. - binds to PtdIns(3)P enriched on early endosomes
BAR domain - binds curved membranes. Most sorting nexins also carry one of these

35
Q

PX domain

A

Binds PtdIns(3)P enriched on early endosomes - sorting nexins have these

36
Q

BAR domain

A

Binds curved membranes - sorting nexins have these

37
Q

Retromer

A

SNX1 and SNX2 are components of this sorting complex. Required for retrograde sorting from endosomes. Return acid hydrolase receptors to the golgi apparatus?

38
Q

Mannose 6 phosphate

A

Sorting signal for soluble lysosomal proteins. Added in the cis-golgi. Terminal modification of high mannose N-linked glycans on lysosomal hydrolases.

39
Q

How do lysosomal enzymes get sorted into the TGN by the M6PR? (Mannose 6 phosphate receptor)

A
  1. M6PR binds to M6P on target lysosomal enzymes in the TGN
  2. DxxLL signal in M6PR cytoplasmic domain is recognized by GGAs in TGN
  3. GGAs recruit clathrin to TGN and form clathrin coated vesicles that include M6PR
  4. CCVs are targeted to late endosomes.
  5. At low pH of late endosomes, MPR affinity for M6P is reduced.
40
Q

What’s another function of MPRs?

A

They retrieve lysosomal proteins that are mistakenly secreted, if AP-2 binds to a YxxL motif.

Cell 600 (9 decks)

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