Sept 22 - Endocytosis and Lysosomal Targeting

Question 1

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

Answer 1

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

Question 2

What is GlcNaC?

Answer 2

N-acetylglucosamine transferase I

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

Question 3

What is the step affected by endo h?

Answer 3

Mannosidase II

Question 4

Three ways that sorting can occur

Answer 4

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

Question 5

Rafeldin A

Answer 5

Collapses multiple compartments into 1

Question 6

Acid hydrolases, cathespins, acid proteases

Answer 6

Proteins that are found in lysosomes

Question 7

Re-derived storage (Glut4) compartment

Answer 7

Retains glucose transporters in insulin-responsive cells

Question 8

List some lysosome related organelles

Answer 8

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

Question 9

What’s the significance of transferrin?

Answer 9

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.

Question 10

Rab5

Answer 10

Enriched on the early endosome

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

Question 11

Rab7

Answer 11

Enriched on the late endosome

Question 12

Things that Rab5 recruits

Answer 12

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

Question 13

PtdIns(3)P

Answer 13

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…

Question 14

PtdIns(4,5)P2

Answer 14

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

Question 15

PtdIns(4)P

Answer 15

Enriched at the golgi, required for secretion.

4 is unlucky, Gollum is also unlucky.

Question 16

PtdIns(3,5)P2

Answer 16

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

3 - endosomal
5 - last number, so late.

Question 17

What does PtdIns(3)P recruit?

Answer 17

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!

Question 18

How do you get internal membranes?

Answer 18

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.

Question 19

How is ESCRT-0 recruited to vacuolar domains?

Answer 19

By binding to PtdIns(3)P

Question 20

Multivesicular body

Answer 20

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.

Question 21

NPVY

Answer 21

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

Question 22

Triskelion

Answer 22

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

Question 23

Traintrack type structures on EM

Answer 23

Associated with clathrin

Question 24

Does clathrin interact with the NPxY signal?

Answer 24

NO there must be other things in between! `

Question 25

AP-2

Answer 25

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.

Question 26

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

Answer 26

COPI at golgi and COPIi at ER exit sites. Cluster of AP-2 recruits clathrin coat, forms pit.

Question 27

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

Answer 27

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

Question 28

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

Answer 28

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

Question 29

What does Shabire mutant tell us?

Answer 29

Drosophila dynamin mutant where endocytic vesicles do not break off

Question 30

What is dynamin

Answer 30

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

Question 31

What do GTPgammaS terminals look like?

Answer 31

GTP gamma S treated nerve terminals look like Shabire terminals.

Question 32

Pinchase

Answer 32

Something that pops off vesicles

Question 33

GGA

Answer 33

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.

Question 34

Sorting nexins

Answer 34

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

Question 35

PX domain

Answer 35

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

Question 36

BAR domain

Answer 36

Binds curved membranes - sorting nexins have these

Question 37

Retromer

Answer 37

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

Question 38

Mannose 6 phosphate

Answer 38

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

Question 39

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

Answer 39

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.

Question 40

What's another function of MPRs?

Answer 40

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