Lecture 4 - Exam 4: Vesicular Trafficking and Autophagy

Question 1

What role does Clathrin play in vesicle formation?

Answer 1

Clathrin plays a structural role in vesicle formation by assembling a basketlike lattice structure that distorts the membrane and initiates budding.

Question 2

What is the formation of clathrin-coated vesicles is regulated by?

Answer 2

A small GTP-binding protein related to Ras and Ran called Arf (also works with COPI).

Question 3

Formation of a Clathrin Coated Vesicle:
How does it work?

Answer 3

1. Arf-GDP is converted to the active GTP-bound form by a Guanine nucleotide exchange factor (Arf/GEF) (GEF switches GDP to GTP)
2. Active Arf-GTP recruits an adaptor protein that “nucleates” both cargo selection and coat assembly.

Question 4

Which is the active form? Which one is the inactive form?
Arf-GDP
Arf GTP

Answer 4

Arf-GDP: inactive
Arf-GTP: active

Question 5

What does the transmembrane receptor select?

Answer 5

Specific cargo proteins

Question 6

Formation of a Clathrin-Coated Vesicle:
Clathrin consists of ________ protein chains that associate with each other to form basketlike lattice structure that distorts the membrane, initiating the bud.

Answer 6

3

Question 7

What is Dynamin?

Answer 7

A large GTP-bound protein that assembles into helical polymers (Think DNA). These constrict when GTP is hydrolyzed.

Question 8

Vesicle Fusion:
What is the mechanism of the first step of vesicle fusion?

Answer 8

Vesicle associated small GTP-binding Rab proteins bind membrane tethering factors. More than 60 different Rab proteins have been identified and different Rab proteins mark different Rab proteins mark different organelles as well as transport vesicles.

Question 9

Vesicle Fusion:
What is the mechanism of the second step of vesicle fusion?

Answer 9

Tethering factors also bind coat proteins and may stimulate formation of complexes between transmembrane proteins called SNAREs (V-SNAREs on vesicles interact with T-SNAREs on target organelles/plasma membrane), and the removal of the coat happens.

Question 10

Vesicle Fusion:
What is the mechanism of the third step of vesicle fusion?

Answer 10

SNARE-SNARE pairing provides the energy to bring the two bilayers close enough to destabilize them and fuse.

Question 11

Is membrane fusion energetically favorable?

Answer 11

NO! Energetically unfavorable.

Question 12

Vesicle Fusion:
SNARE proteins have what kind of domain? What does this allow? What step does this take place in the vesicle fusion process?

Answer 12

Coiled-coil domain.
It provides the energy to get the plasma membranes to fuse (which is energetically unfavorable). More specifically, this allows the domain to bind to other coiled-coil domains and zips the SNAREs on vesicle and target membranes together.
This brings the 2 membranes into direct contact and leads to fusion of the lipid bilayers.
Still on the third step.

Question 13

The SNARE proteins have a coiled-coil domain that is similar to?

Answer 13

Nuclear lamins

Question 14

What is a coiled-coil?

Answer 14

A structural motif in proteins in which 2-7 alpha-helices are coiled together like the strands of a rope.

Question 15

What are the main roles of lysosomes?

Answer 15

Acts as the waste disposal system of the cell by digesting unwanted materials. They can break all kinds of polymers - proteins, nucleic acids, carbohydrates, and lipids.
They serve to degrade material taken up from the outside of the cell as well as obsolete cellular components.

Question 16

What is the lysosome’s shape?

Answer 16

Spherical, but shape can vary depending on the materials that have been taken up for digestion.

Question 17

What identifies lysosomal membranes?

Answer 17

LAMP1 (Membrane protein)

Question 18

What is the pH of a lysosome?

Answer 18

Very acidic, pH 4.5-5

Question 19

How many different degrative enzymes do lysosomes have?

Answer 19

60

Question 20

Will lysosomal enzymes work in the cytosol?

Answer 20

NO! most lysosomal enzymes will not work in the cytosolic pH of 7.2

Question 21

What does whole Immunohistochemistry (AKA antibody staining) tell you?

Answer 21

Can tell you when and where proteins are expressed in a cell/tissue/embryo.
It also tells us where in an individual cell proteins are located (also known as sub-cellular localization).

Question 22

How does Immunohistochemistry (aka antibody staining) work?

Answer 22

Primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabeled).
Secondary antibodies are raised against immunoglobulins of the primary antibody species. The secondary antibody is usually conjugated to a linker molecule.