VL 35 (Michael Sauer)

Question 1

Vacuoles of plants

Answer 1

Question 2

Soluble proteins made in the ER – bulk flow:

Answer 2

• No sorting signals – secreted into apoplast by default secretory pathway

Question 3

Vacuolar sorting signals and receptors:

Answer 3

VSS
* required for VSR-recognition o groups
▪ sequence-specific vacuolar sorting signal (ssVSS): consensus sequence
▪ C-terminal vacuolar sorting signal (ctVSS): hydrophobic aa

VSRs
* function: sorting, packaging of soluble vacuolar proteins into transport vesicles o bind cargoes in donor compartment → release in acceptor compartment
* cargo binding enhanced by high c(Ca2+)
* structure
▪ N-terminal luminal domain: bind cargo
▪ Transmembrane domain
▪ C-terminal cytosolic tail: interaction with trafficking components for vesicle
formation + own recycling

Question 4

Receptor + ligand are recruited into clathrin coated
vesicles

Answer 4

Question 5

Receptor retrieval by the retromer complex

Answer 5

• membrane protein transport in vesicles between endosomes – TGN
• mediate retrieval of various transmembrane receptors

Question 6

Summary and alternative view:

Answer 6

Question 7

How to degrade transmembrane proteins? – MVB/Prevacuolar Compartment

Answer 7

Late endosomes (LE)/Multivesicular Bodies (MVB) contain intraluminal vesicles (ILVs)

1. endocytosis:
* ligand binds mytogene (EGF)
* dimerization
* switching-off signaling by receptor-mediated endocytosis via clathrin-coated vesicles
2. gathering:
* CCV-transport to endosomes
* interaction: clathrin - cytosolic receptors
3. concentration:
* receptor concentration at the membrane
* Ub-labeling interaction of receptors with ESCRT complex
* ESCRT complex assembly by PIP3
4. budding inward:
* bulge formation in the endosome (away from cytoplasm)
* membrane fusion
* vesicle formation in endosome
* = MVB
5. fusion MVB lysosomes:
* hydrolases released into endosome→degradation (vesicles with EGF receptor)

Question 8

ESCRT complex generates intraluminal vesicles:

Answer 8

➔ MVB-formation
1. Mono-Ub membrane protein in EE binds ESCRT-0 (=HRS) binding to PIP3 at
the endosomal membrane (via PH domain)
2. clathrin & ESCRT-0 concentrate mono-Ub proteins in clathrin-coated
domains
3. ESCRT-I, -II, -III recruitment to cytosolic membrane side; binding: ESCRT proteins to Ub and PIP3; ESCRT-III polymerizes formation of filamentous
helices
4. ATP hydrolysis by AAA-ATPase: Vps4 triggers membrane remodeling, constriction and disassembly of helical complexes

Question 9

Phosphoinositides (PIPs):

Answer 9

Question 10

Phosphoinositides mark different membrane domains

Answer 10

Question 11

Autophagy – Degrading protein aggregates and organelles:

Answer 11

Question 12

Steps of Autophagy

Answer 12

= process by which cell break down and utilize their own components
Phases
* Initiation: three At9-vesicles
* Phagophore (double-membrane) nucleation
* Elongation and autophagosome formation: membrane-addition +
cargo internalization; fusion of phagocytic cup opening →
autophagosome
* autophagosome-vacuole (lysosome in mammalian cells) fusion o Cargo degradation: inner membrane + content are hydrolyzed
* autophagosomes take up cellular material (e.g. misfolded proteins/whole organelles to be degraded); fuse with lysosomes→autophagolysosomes