Lecture 11 - endomembrane system part 6

Question 1

What is a lysosome?

Answer 1

vacuole in plants, digestive organelle
contains 60 different soluble acid hydrolase enzymes

Question 2

What are the functions of lysosomes?

Answer 2

degrades all types of macromolecules
also plays key role in degradation of larger cellular components/organelles - autophagy

Question 3

When are lysosomes soluble acid hydrolase enzymes enzymatically active?

Answer 3

low pH 4.6 of lumen (maintained by membrane bound ATPase proton pumps), resident lysosomal membrane proteins protected from degradation by attached lumen facing carb groups

Question 4

Where are the products of lysosomal degradation transported to?

Answer 4

cytoplasm - reused by various biosynthetic metabolic pathways

Question 5

What do the ATPase proton pumps in the lysosome pump?

Answer 5

H+ from cytoplasm into lumen of lysosome

Question 6

How are proteins trafficked to the lysosomes?

Answer 6

synthesized and initially N-glycosylated in RER then transported from ERES to golgi
in cis cisternae, glycosylated lysosomal destined proteins are further modified
proteins with an M6P signal patch are destined to the lysosome

Question 7

When does the biosynthetic pathway interact with the endocytic pathway?

Answer 7

at late endosomes, responsible for uptake of pm proteins/receptors and extracellular materials

Question 8

What are the steps to trafficking of lysosomal proteins out of the TGN?

Answer 8

1. in TGN soluble M6P bearing lysosomal destined proteins recognized by M6P receptor
   - integral transmembrane protein
2. M6P receptor mediates subsequent concentration of soluble lysosomal proteins into nascent clathrin-coated transport vesicles
   - membrane cargo receptor proteins at ERES involved in binding soluble proteins to be packaged and transported in COPII coat vesicles for golgi
3. cytoplasmic-facing domain of M6P receptor binds to AP1 and GGA adapter coat proteins
   - AP1GGA mediate vesicle cargo section, also serve as linker for clathrin coat vesicle assembly
4. recruitment of AP1GGA adaptor proteins from cytoplasm to TGN surface mediated by GTPase Arf1
   - Arf1 binding to GTP causes conf. change initiates outward bending of membrane
5. AP1/GGA proteins recruited by Arf1-GTP also bind to major component of vesciel but coat clathrin
6. triskelions recruited from cytoplasm self assemble to form outer scaffolding of coat in growing vesicle
   - inner layer consist of AP complex linked to Arf1 and M6P receptor
7. release of clathrin vesicle from TGN mediated by dynamic
   - dynamic recruited from cytosol to connected between growing clathrin coated bud and TGN membrane
   -dynamin proteins self assemble to form dynamic ring
8. GTP hydrolysis causes conf. change in dynamic ring resulting in twisting and pinching off of vesicle
9. after pinching off the clathrin coat disassembles
   - Arf1 GTP -> Arf1 - GDP
   - Arf1-GDP, AP complex and clathrin triskelions released into cytoplasm
10. nascent vesicle with M6P receptor bound soluble lysosomal cargo proteins fuses with late endosome
    - mediated by Rab/Rab effector and SNARES
11. acidic interior of late endosomes causes M6P receptors to dissociate from proteins
    - phosphate removed from M6P groups in proteins prevents rebinding, recycled
12. retromer complex coat assembles on cytoplasmic surface of late endosome, retromer coat disassembles after vesicle formation at late endosome
13. retromer vesicles also traffic to pm through receptor mediated endocytosis
14. late endosome fuses with lysosome (mediated by Rab and SNARES)
    - late endosome luminal contents released into lysosome interior (lysosomal proteins activated by pH)
    - materials from endocytic pathway also delivered to lysosome for degradation

Question 9

What are AP1GGA adaptor coat proteins?

Answer 9

cytoplasmic proteins with multiple protein-protein binding domains

Question 10

What is Arf1

Answer 10

equivalent to sar1 in COPII coat at ERES

Question 11

What does clathrin consist of?

Answer 11

three light chain polypeptides and three heavy chain polypeptides, forms a three-legged structure, triskelion which for scaffolding of clathrin-coated vesicles

Question 12

How do triskelions form on the surface of the clathrin vesicle?

Answer 12

intially self assemble to form hexagons that lie flat on membrane, they then assemble to form pentagons (this causes further curvature)

Question 13

What are late endosomes the junction of?

Answer 13

biosynthetic and endocytic pathways
- involves early endosomes, late endosomes and ESCRT machinery

Question 14

what is delivered to endosome/lysosome for degradation?

Answer 14

materials derived from pm and extracellular space via receptor mediated endocytosis