Lecture 3

Question 1

Vesicle Fission (Scission)

Answer 1

Budding Out of membrane
-initiated at luminal side (HP)

Question 2

Vesicle Fusion

Answer 2

Budding in
-initiated at cytosolic side

Question 3

Clathrin-Coated Vesicles

Answer 3

-Endocytosis
-bud from Plasma Membrane and TGN
-lattice-like surface
-much sturdier

Question 4

Clathrin

Answer 4

-Assembly unit is Triskelion
-can spontaneously associate into an empty cage, however
-has adaptors (AP) and accessory proteins

Question 5

AP (assembly proteins)

Answer 5

-clathrin adaptors
-mickey mouse
-promote assembly
-Adaptor binds to tyrosine-based signals (Yxxf) in tails of receptors

Question 6

Coincidence Detection

Answer 6

multiple weak interactions cooperate to ensure specific membrane targeting
EX: APs require binding of lipid and cargo

Question 7

GTPases

Answer 7

-regulate vesicle formation
-gives energy for directionality and accuracy

Question 8

Arf1

Answer 8

GTPase for COPI assembly, also needed for clathrin-coated vesicles

Question 9

Sar1

Answer 9

GTPase for COPII assembly

Question 10

Formation of Clathrin-coated vesicles

Answer 10

Arf1 recruits AP-1 and GGA adaptors

Question 11

GGA

Answer 11

-Single-unit clathrin adaptors
-VHS domain that binds with sorting receptor
-GAT domain binds with Arf1
-Hinge binds with Terminal domain of clathrin
-Ear binds with accessory proteins like AP-1

Question 12

Dynamin

Answer 12

-GTPase that acts as a pinchase and drives scission
-polymerizes and tightens
-Clathrin coated vesicle

Question 13

Sar1/Arf1

Answer 13

acts as pinchase for COPI and COPII vesicles

Question 14

Uncoating of Clathrin-Coated Vesicles

Answer 14

-ATP-dependent
-Hsc70 (cystolic form) with Auxilin as its J-domain

Question 15

COPI-coated vesicles

Answer 15

-carry between cisterna of Golgi and Golgi to ER
-Arf1 GTPase and 7 coatemers are needed
Arf1-ADP is converted to Arf1-ATP by and exchange factor which recruits Arf1 to membrane
-hydrolysis of Arf1 leads to uncoating
-its dimerization drives COPI scission

Question 16

Brefeldin A (BFA)

Answer 16

-inhibits exchange factor from recruiting COPI to membrane of donor

Question 17

COPII-coated vesicles

Answer 17

-ER to Golgi
-Sar1 GTPase with 2 heterodimeric coat complex
-Exchange factor that phosphorylates GDP to GTP is Sec12
-has a lot of holes and the inside can interact with the outside

Question 18

ERES (ER Exit Sites)

Answer 18

formation of collar at base of tubules from ER allows exit of larger proteins

Question 19

Fluorescent Proteins

Answer 19

mostly derived from aquatic animals (Jellyfish)
4 problems
1) Oligomerization-dimerizes weakly
2) Cytotoxicity - proteins aggregates and be toxic
3) Slow Maturation - WT dsRed 12h halftime
4)Photobleaching and Photoconversion
- bleach fast and red turns green