intracellular membrane trafficking

Question 1

How do vesicles generically work? what do they transport?

Answer 1

vesicles bud form the donor compartment and fuse with the target compartment. Vesicles transport cargo including lipid bilayers, bilayer-associated proteins, and soluble proteins

Question 2

What are the three pathways used by vesicles?

Answer 2

Biosynthetic-Secretory pathway
Endocytic pathway
Retreival pathway

Question 3

what are coat proteins? what is their function?

Answer 3

coat proteins enable vesicle budding form donor compartments.
Function:
Induce vesicle buddig
recruit soluble and TM cargo proteins to vesicle
they are rapidly shed from vesicles after budding
remain in vicinity of donor and are reused
may form cage like structures (clathrin)

Question 4

Three best characterized coat proteins

Answer 4

1. Clathrin: The PM and trans-Golgi uses
2. COPI: the cis-golgi uses
3. COPII: the ER uses

Question 5

why is it important to have different coat proteins for different organelles?

Answer 5

because different coat proteins recruit different cargo molecules and thereby control cargo identify and contents

Question 6

General steps for Clathrin, COPI and COPII

Answer 6

the donor buds as the ligand binds to the receptor. coat proteins form the bud. v-SNAREs and Rab are on the vesicle membrane as fission occurs and the vesicle containing cargo is released. Rab hydrolyzes GTP to GDP and coat proteins release to be recycled. The vesicle docks as its v-SNAREs entangle with membrane t-SNAREs and bring the membranes close enough to fuse.

Question 7

What is the structure of Clathrin?

Answer 7

a triskelion with three heavy chains and three light chains. this structure allows spontaneously formed coat structures

Question 8

what are Adaptins?

Answer 8

Clathrin binds to Adaptins, not to the bilayer directly.
Adaptins are adaptor proteins, they position themselves between the vesicle bilayer and the clathrin coat. They bind to the cytosolic domains of TM cargo receptor proteins and phosphoinositides.
Each variety of adaptin protein specifically binds to a different set of cargo receptors

Question 9

Steps in the formation of clathrin coated vesicles

Answer 9

1. transmembrane cargo receptors bind specific cargo molecules and thereby recruit them to the vesicle and hold them inside
2. cytosolic adaptor proteins (Adaptins) bind cytosolic domains of TM cargo receptors and phosphoinositides
3. Adaptins recruit clathrin to cytosolic surface of vesicle
4. binding of clathrin induces bilayer curvature, encourages budding
5. both clathrin and adaptin dissociate from vesicle shortly after budding, remaining near donor for recycling

Question 10

How is AP2 (Adaptin protein 2) a good example of specificity?

Answer 10

AP2 can only bind to the phosphoinositide PIP2. it is in a locked conformation until it binds PIP2 and the correct cargo receptor. This is specificity because AP2 will only form vesicles in membranes that have PIP2, which will only occur in specific areas.

Question 11

what enables scission of clathrin coated vesicles?

Answer 11

Dynamin: a monomeric G-protein that constricts the neck of budding vesicles. It recruits other proteins to the vesicle neck and causes the vesicle to pinch off

Question 12

what can mutations of dynamin cause?

Answer 12

either enhanced or inhibited vesicle scission. In Drosophilia the mutant shibire causes no scission to occur at PM vesicles, so recycling of exocytosed synaptic vesicle is greatly reduced and fly is paralyzed. clathrin coated pits have tube like structure due to failure of scission

Question 13

what is retromer and what is its structure?

Answer 13

the 4th coat protein. composed of several subunits.
BAR domain: mediates dimerization of retromer and attachment to curved bilayers (only attaches to curved bilayers)
PX domain: binds directly to PIP3

Question 14

What does retromer do? where is its pathway?

Answer 14

retromer assembles on Endosomes. It buds vesicles that enter a retrieval pathway.
Pathway returns cargo to the Golgi. cargo includes TM cargo receptors for acid hydrolases and lipid bilayer

Question 15

what is a coincidence detector? what’s an example?

Answer 15

a detector that responds to two or more simultaneous conditions. Retromer is a coincidence detector as it binds to Endosomal bilayers ONLY when these three conditions occur:

1. retromer binds the cytoplasmic tails of cargo receptors
2. retromer interacts directly with a curved bilayer
3. retromer binds the PI(3)P present on endosomes

Question 16

why are phosphinositides phosphorylated?

Answer 16

each phosphoinositide can by phosphorylated at a different position on the sugar ring, each phosphorylation brings a negative charge. Different shapes and charges of head groups influence what can bind it. Different bilayers have different phosphoinositides which affect what can bind it