Lecture 3

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

Budding in -initiated at cytosolic side

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

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

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

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

-regulate vesicle formation -gives energy for directionality and accuracy

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

GTPase for COPII assembly

Arf1 recruits AP-1 and GGA adaptors

-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

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

acts as pinchase for COPI and COPII vesicles

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

-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

-inhibits exchange factor from recruiting COPI to membrane of donor

-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

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

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