Lecture 6: Vesicular transport continued.

Vesicle coats by emphasizing clathrin-coated vesicles in endocytosis, Vesicular transport & fusion: Rab GTPase and SNARE proteins, organelles for macromolecule degradation

Trans Golgi Network (TGN): protein sorting station

goal: recognizes sugar enzyme to leave the trans Golgi for transport to Lysosome
1. Signal-Mediated Diversion to LYSOMES (via Endosomes)
- mannose-6-phosphate receptor

2. Signa-mediated diversion to secretory vesicles (for regulated secretion)

   • no signal, bulk flow for exocytosis

3. Constitutive Secretory Pathway
   

clathrin-coated proteins

mediate transport originating from the Golgi apparatus, endosome, and plasma membrane

COPII-coated proteins

anterograde
From ER → Golgi

COPI-coated vesicles

retrograde
From Golgi → ER

Retromer

froms coats on transport vesicles for a retrieval pathway from endosomes to Golgi apparatus

The most abundant type of vesicle coating is _______, which is found in the trans Golgi.

clathrin

Clathrin is composed of

3 heavy chains and 3 light chains assembled into the triskelion

Clathrin triskelion

basketlike framework of hexagons and pentagons on the cytosolic surface of membranes
- INDUCES the formation of coated buds, which eventually pinch off to become clathrin-coated vesicles.

Adaptor proteins (adaptins)

another major coat component in clathrin-coated vesicles, form a discrete inner layer of the coat, positioned between the clathrin cage and the cytosolic face of the membrane
- bind to clathrin coat (heavy & light chains) and recruit it to the membrane surface where it assembles and BENDS the membrane
- NEEDS cargo to be activated

Endocytosis

clathrin-coated vesicles formed at the plasma membrane, and deliver their cargoes to endosomes

Uptake of LDL particles

receptor-mediated endocytosis to import LDL particles
- 1 in 500 inherits 1 defective LDL receptor genes
- Individuals with defective receptors only have high blood cholesterol levels-predispose to atherosclerosis
Target is LYSOSOMES

A _________ recognizes lysosomal hydrolases in the Golgi apparatus.

GlcNAc
N-acetylglucosamine

Mannose-6-phosphate is a

lysosomal protein “marker”
(put into vesicles arriving in lysosomes)

Inclusion-cell (I-cell) disease

mucolipidosis II: patient cells cannot carry the mannose-6-phosphate modification of lysosomal enzymes
Result: blood accumulates in red blood cell

The protein that is responsible for pinching off clathrin-coated vesicles by GTPase protein is

dynamin
Mechanism: GTP HYDROLYSIS causes conformational change

shibire mutant

gene encodes the GTPase dynamin, creates pits but NO pinching off the vesicle

Soluble ER proteins are retrieved from…

the Golgi by COPI-coated vesicles

What gene sequence allow protein in stay in the ER?

KDEL - C terminus

Vesicular transport takes place between different _______ of Golgi, as demonstrated by experiments using Golgi apparatus isolated from two different cell types (mutant golgi with virus and nomral golgi. Mutant cells lack the enzyme _________. And requires ______ & ________.

cisternae
GlcNAc-transferase I
ATP & Cyotsol

How does vesicle know when to be integrated into target membrane?

SNARE and RAB GTPase
1. Activation of Rab by its specific GEF
2. Interaction between Rab-GTP and its effector-tethering
3. Vesicle docking
4. Vesicle fusion
5. ~60 Rab GTPase proteins in human

______ Rab GTPase proteins in human

60

Rab GTPase

inserted into the lipid bilayer (C-terminus)
triggers the recognition of vesicles with the destination membrane
allows tethering to Rab effector for docking attached to target membrane

SNARE protein

has coil-coil domains to facilitate fusion of vesicle membrane & target membrane
starts fusion and releases Rab-GDP

Vesicle fusion with target membranes (synaptic vesicles and exocytosis)

1. Vesicle docking via Rab-GTP and its effector

2. Formation of SNARE complex: specific interaction between v-SNARE and t-SNARE via their coil-coil domains

3. Arrival of Ca to open fusion pore

4. Vesicle fusion complete and the release of the cargo molecules

   1. *Distinct pairs of v-SNARE and t-SNARE at donor and destination compartments

Homotypic vesicle fusion

takes place for ER-generated vesicles and for producing larger endosomes from small ones

What enzymes are involved in homotypic vesicle fusion to make larger endosomes?

NSF + ATP
released v-SNARE and t-SNARE together to bind to another endosome

Insulin-regulated exocytosis for glucose uptake

1. Presence of glucose makes insulin bind to receptor on membrane.

2. Intracellular signal causes relocalization of glucose transporter to PM to BOOST glucose uptake into the cell.

3. Cell takes in more glucose for exocytosis

In plants, Lysosomes, coming from ______, digests broken organelles. Therefore, a _____ pump that requires _____ is needed to make an ______ environment for acid hydrolases.

TGN
H+
ATP
acidic

What is the plant cell’s counterpart for lysosomes?

vacuoles

Organelles involved in macromolecules, pathogen & dysfunctional organelle degradation:

• Early endosomes

• Late endosomes

• Phagosomes

• Autophagosomes

• Lysosomes/vacuoles

Autophagy

unwanted organelles are engulfed by autophagosomes and acidic ally hydrolysis by lysosomes.

Acrosome

lysosome enzyme that breaks down the membrane of sperm for fertilization