Intracellular Compartments and Vesicular Trafficking

Question 1

What are the two directions that vesicles can move from the ER?

Answer 1

Anterograde: from the ER to Golgi, plasma membrane and to lysosomes

Retrogrades: From endosomes to golgi to ER

Question 2

Is the inside of vesicles more similar to the cytosol or extracellular space?

Answer 2

Vesicle interiors are topologically the same as extracellular space

Question 3

What are the 5 major steps of vesicular transport?

Answer 3

1) Cargo selection/loading
2) Vesicle budding
3) Vesicle transport
4) Vesicle targeting
5) Vesicle fusion/ cargo delivery

Question 4

How does the clathrin coat impact the membrane shape?

Answer 4

The binding of Clathrin induces a vurvature into the membrane which begins the formation of a transport vesicle

Question 5

True or false: Vessicles passively move throughout the cytosol.

Answer 5

FALSE

Vesicles are relatively large and require active transport by motor proteins

Question 6

What is the function of Rab proteins?

Answer 6

Rab proteins connect transport vesicles to motor proteins

Rab proteins also target vesicles to the appropriate destination point and interact with Rab effectors (tethering proteins) once they are near the target membrane

Question 7

What are the three different coat proteins and when does each play a role in vesicle transport?

Answer 7

COPI: Retrograde movement

COPII: Anterograde movement

Clathrin: Exo/Endo-cytic vesicles between golgi, endosomes and plasma membrane

Question 8

What role does Sar1 play in the formation of vesicles?

Answer 8

Sar1 providees a site to which COPII coat proteins can bind (Coat recruitment GTPase)

Sar1 is activated by Sar1-GEF which exchanges GDP for GTP.

The Sar1-GTP complex is membrane bound and marks the transitional ER for COPII binding

Question 9

What amino acid sequence is the “ER retention motif”?

Answer 9

KDEL

This sequence is unique to proteins that should remain in the ER lumen. If the protein is accidentally sent to the golgi network, it will be returned via retrograde trafficing

Question 10

How are specific cargo molecules selected for export in vesicles?

Answer 10

Receptors specific to the cargo molecules collect the cargo at the membrane and recruit adaptor proteins and clathrin coat proteins for vesicle formation

Question 11

What are the different mechanisms by which integral membrane proteins can be selected for transport?

Answer 11

The cytoplasmic tails of the receptors are marked via:

Phosphorylation

Amino acid motifs

Ubiquitination

Question 12

Describe how LDL receptors make cholesterol available to the cell.

Answer 12

LDL, a lipoprotein complex used to transport cholesterol between cells, binds to LDL receptors on the cell surface

Endocytosis via clathrin coated vesicles is initiated

Clathrin leaves and acidic endosomes are formed

LDL is hydrolyzed in lysosomes and cholesterol is made available to the cell

Question 13

What is the result of a mutation that prevents LDL receptors from binding to clathrin adaptor proteins?

Answer 13

“Hypercholesterolemia”

Less cholesterol can be uptaken into the cell

Most dangerous clinical result: atherosclerosis

Question 14

Describe the structure of clathrin

Answer 14

Triskelion structures with 3-fold symmetry

Clathrin molecules self associate into icosahedrons

Question 15

What does Dynamin do?

Answer 15

Dynamin is a snake-like protein that helps with the vesicle fission process

ATP hydrolysis tightens the dynamin coil which “pinches off” the budding vesicle

Question 16

Do coat proteins impact vesicle targeting?

Answer 16

No. Coat proteins are removed after the budding stage.

The specificity of vesicular transport is determined by entities below the coat proteins on the vesicles

Question 17

What role does phosphatidyl inositol play in vesicular targeting?

Answer 17

Phosphatidy inositol has 5 OH groups that can be selectively phosphorylated

The phosphorylation pattern dictates which proteins can bind and thus the destination of that vesicle

Question 18

What is a Rab5 membrane domain?

Answer 18

Markings on target membranes (and vesicular surfaces) are built up to encourage specific vesicle targeting/recognition

These domains are made up of active Rab5-GTP, Rab effector proteins, and phosphatidyl inositol

Question 19

What are the SNARE proteins?

Answer 19

Proteins that promote vesicle fusion by positioning the vesicle close to the membrane.

v-SNARE (on vesicules) interacts with t-SNARE (target membrane)

When bound: 4 stranded alpha helical coiled coil

Question 20

How are SNAREs taken apart following vesicle fustion?

Answer 20

They must be pried apart by accessory proteins and NSF which require ATP energy

Question 21

How does BOTOX work?

Answer 21

Botulism toxin binds to neurons at neuromuscular junctinos and cleaves SNAREs resulting in paralysis because acetylcholine vesicles cannot fuse

Question 22

Describe the structure of the golgi apparatus.

Answer 22

Organized into 5 different compartments

cis Golgi network, cis cisterna, midial cisterna, trans cisterna, and trans Golgi network

Question 23

What are the two pathways of protein secretion from the Golgi appartus?

Answer 23

Constitutive secretory pathway: vesicle fusions are unregulated

Regulated secretory pathway: do not fuse without an extracellular signal

Question 24

How are secretory storage granules formed?

Answer 24

Forward transport moves cargo into vesicles while reverse transport removes unintended cargo from the vesicles

Increasing concentrations of homogenous cargo are formed by this process