Lecture 12 Flashcards
What proteins are involved in vesicles knowing where to go?
Rabs and SNAREs
What are Rabs?
Large family of GTPases that line cytosolic surface of membranes
- Only certain Rabs are on specific membrane compartments
What happens when Rabs are inactive?
They are GDP bound
- Rab-GDP associates with the dissociation inhibitor (GDI) and stays soluble in cytoplasm
What happens when Rabs are active?
They are GTP bound
- Rab-GTP associates with membrane and interacts with Rab effectors (assist with membrane fusion and tethering)
How many different Rabs have been found in human cells?
70
What do Rab effectors comprise of?
Many different types of proteins (motor and tethering proteins)
What do Rab-GTP and effectors form?
Multi-protein complexes that form discrete membrane patches capable of receiving specific vesicles or budding new vesicles
Where is the subcellular location for Rab5?
- Early endosomes
- Plasma membrane
- Clathrin-coated vesicles
Where is the subcellular location for Rab7?
Late endosomes
What has to occur in the ER when proteins are being transported to the Golgi in transport vesicles?
Quality control to ensure proteins are properly folded
- In some cases, 90% of the protein is degraded in lysosomes and only 10% makes it to the final location (e.g., acetylcholine receptors)
What participates in quality control?
Chaperones
- Some proteins bind to chaperones in the ER to ensure protein structure is maintained
What happens to vesicles and vesicle tubules?
Diffuse through cytoplasm or follow cytoskeletal tracts
- Microtubules and actin are used
What are SNAREs?
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor
- Required for membrane fusion
- Catalyze membrane fusion and provide binding specificity
- Over 35 types in mammalian cells
What types of SNAREs are there?
v-SNAREs and t-SNAREs
What are v-SNAREs?
Single polypeptide chains found on vesicle membranes
- e.g., synaptobrevin
What are t-SNAREs?
Two or three polypeptide chains found on target membranes
- e.g., SNAP25
What do v-SNAREs and t-SNAREs do?
Interact and “lock” membranes together
- Forms a four helical bundle
What does synaptotagmin do in a v-SNARE/t-SNARE complex in neuronal secretory vesicles?
Serves as a Ca2+ sensor to activate exocytosis
Why do SNAREs have to dissociate?
To enable further rounds of fusion
What is the key regulator in SNARE disassembly?
NSF
What is NSF?
ATPase that uses ATP to unravel the SNARE complexes
What are lysosomes?
Single membrane-enclosed organelles containing hydrolytic enzymes (acid hydrolases) that degrade proteins
Why is pH important for lysosomes?
Enzymes have to be proteolytically cleaved to be activated and need acidic pH (4.5-5) for optimal activity
How do lysosomes maintain acid pH?
They have a vacuolar ATPase to pump H+ into the lumen
How are lysosomes stained by lead phosphate?
Acid phosphatase and glutaraldehyde
How are lysosomes supplied with enzymes?
Lysosomes fuse with other vesicles that come from endocytosis (endosome) and form vesicles from Golgi (from TGN)
How are proteins destined for the lysosome recognized in the Golgi?
A mannose-6-phosphate (M6P) group covalently attaches to lysosomal hydrolases in cis-Golgi
What do M6P receptor proteins do?
M6P receptor proteins present in trans-Golgi bind to M6P-containing protein in the lumen of TGN
- Only bind to M6P at pH > 6.5 (pH in early endosomes)
What happens as pH drops during endosome maturation?
M6P-containing proteins are released from the receptor and the phosphate group is cleaved by phosphatase
What are some lysosomal storage disorders (LSD) due to?
Excess cholesterol in the membrane which locks SNARE complexes and prevents dissociation
- Some due to deficiencies in acid hydrolases or other enzyme components of the lysosome
How many genetic (autosomal recessive) LSDs are recognized in humans?
50
Who is affected by LSDs?
Mostly affects children and survival is short (months to a few years old)
What is an example of an LSD?
Tay-Sachs disease
What are characteristics of Tay-Sachs disease?
- Mutation in HexA gene (4 bp insertion)
- HexA encodes hexosaminidase A which degrades sphingomyelin in lysosomes
- 1 in 2,500 births in Ashkenazi Jewish population (1 in 30 are carriers)
- Children die by 4 years of age
