Lecture 14: Trafficking and the Endomembrane System II- Secretory and Endocytic Pathways for Protein Sortingo Vesicles transport membrane and soluble proteins from one membrane-bound compartment to anothero Transport vesicles Collect cargo proteins in membrane budding and deliver to next compartment via fusion with target membraneo Secretory pathway Distribution of soluble and membrane proteins from ER to final destinations at the cell surface- Synthesis of proteins in ER (signal/targeting sequence)- Protein traffickingo Endocytic pathway Vesicles bud from plasma membrane take up soluble extracellular proteins and deliver them to lysosomes via late endosomes- Kinetics of transport from ER-Golgi-plasma membraneo Viral membrane glycoproteins  synthesized and inserted into host cell membrane by constitutive secretory pathway- Transport from ER to Golgi: Measuring Glycosylationo Detecting secretory protein carbohydrate modification by compartment-specific enzymes elucidates trafficking through different compartments o Protein movement to cis-Golgi and modification Protein retained in ER  not modified Protein moved from ER to cis-Gogli  core oligosaccharide Man8 is trimmed to Man5 by cis-Golgi resident enzymes- Endoglycosidase D  cleaves oligosaccharide chains from proteinso Yeast sec Mutants Identified 5 stages in secretory pathway RELOOK LATERMechanisms of Vesicle Budding and Fusion- Vesicle budding from donor membraneo Vesicles bud from donor membrane and fuse with target membraneo Vesicle budding from donor membrane Recruitment of GTP-binding G protein Cytosolic coat protein complexes bind domain of membrane cargo protein Coat binding evaginates membrane Some cargo proteins act as receptors to bind soluble proteins in lumen and capture them into budding vesicle Donor membrane-specific SNARE proteins captured in budding vesicle membrane Donor membrane fusion  pinches off coated vesicle Coated vesicle uncoated in cytosol  uncovers projecting v-SNARES for fusion targeting- Vesicle fusion with target membraneo Vesicles bud from donor membrane and fuses with specific target membraneo Assembly of protein coat drives vesicle formation and selection of specific cargo moleculeso Vesicle fusion with target membrane Targeting  interaction of specific v-SNARE with specific target membrane t-SNARE- Coated Vesicle Involved in Protein Traffickingo 3 major types of transport-specific coated vesicles  each with different ype of protein coat Conserved set of monomeric GTPase proteins control assembly of different vesicle coats- Clathrin, COPI, and COPII- GTPases Initiate Coat Assemblyo Two conformations of GTPase  GTP bound and GDP bound GTP bound = active conformation  can interact with target proteins GDP bound = inactive conformationo GEP  stimulates replacement of the bound GDP with a GTP (off  on)o GAP  stimulates GTP to GDP (on  off)- Sar1: Assembly and Disassembly of COPII