Lysosome (Martens)

Question 1

What is the role of lysosomes? Name a few specific examples.

Answer 1

Interaction with many structures in cell:
- bulk degradation
- nutrient sensing
- signal integration
- transport

Examples:
Mit:
- determines fision sites for mitochondria
- metabolite transport
ER:
- interacts with ER which determines fission sites of lysosomes
- cholesterol sensor and lipid transport
Phagosome:
- pathogen defense -> fusion
Autophagosome:
-self-eating (starvation, recycling) -> fusion
Late endosome:
-endocytosis of PM/extracellular material -> fusion
Retrograde transport

Question 2

What importat enzymes maintain the physiological function of lysosomes? Name a few.

Answer 2

Lumenal:
- lipases
- proteases
- DNAses
- glycosidases
- nucleases

Membrane bound:
- V-ATPase H+ pump
- ion transporters (Fe+, Ca2+, Cu2+…)
- AA transporters

Question 3

How are proteins destined for lysosomes recognized?

Answer 3

Mannose 6 phosphate (M6P):
lysosomal hydrolase has M6P attached to N-glycan -> transporting signal to be recognized

**In Golgi: **
1) LH’s signaling patch is recognized by GlcNAc phosphotransferase
2) N-glycan carrying terminal mannose is is bound together with UDP-GlcNAc in the catalytic site
3) P-GlcNAc is donated from UDP-GlcNAc
4) GlcNAc is removed.

Question 4

How is the M6P-bound cargo translocated into a lysosome?

Answer 4

M6P-M6P receptor with adaptors:
1) ARF-GDP (soluble) + ARF-GEF
2) ARF-GTP (via myristoylated dom.) bound to TGN
3) GGA clathrin adaptor (Golgi-localized gamma-ear-containing Arf-binding protein) colocs with M6PR-M6P-cargo
4) Translocation

Question 5

How is the M6PR retrieved back to TGN?

Answer 5

Early endosomes: acidic pH -> dissociation + M6P removal -> components are retrieved via retromer coats back to TGN

Retromer coat: cargo (e.g. M6P inside) bound to VPS35. Other important proteins: VPS29, VPS26, SNX dimers bound via PI3Ps

Question 6

How are proteins endocytosed? What is their usual path? How are the individual compartments distinguished?

Answer 6

PM -> clathrin coated endocytic vesicle -> early and late endosome -> multivesicular body -> lysosomes

Rab GTPase distinguisment of compartments:
- early: Rab5
- late: Rab7

1) Rab5-GDP + Rab5-GEF
2) Rab5-GTP (via myristoylated dom.) bound to early endosome
3) Rab5-GTP activates PI3K
4) PI2P -> PI3P
5) PI3P recruit Rab5 effectors
—
Handover to late endosome:
6) Rab5-GTP recruits Rab7-GEF + EEA1 (tethering molecule) + PI3K -> Rab7-GTP
7) Rab7-GTP recruits Rab5-GAP -> Rab5-GDP

Question 7

Name a specific receptor that can be endocytoses and explain the pathway. Briefly explain the effector ESCRT complex. What other functions does it fulfil?

Answer 7

EGFR: lysis in lysosome
1) EGFR dimer is ubi
2) Ubi recognized by Epsin and Eps15
Epsin: membrane-binding dom., ubi-binding dom., clathrin-
binding dom.
3) Clathrin-coated endocytosis
4) PI3P-EGFR is recognized by ESCRT
5) ESCRT1 -> ESCRT2 -> ESCRT3 maturation
6) Inward budding and scission of the vesicle

Other ESCRT proteins: Gag, Alix, Vps4

Other functions:
- PM repair
- exocytosis
- HIV budding (ble)