Endomembranes I: Moving Proteins and Membranes in the Secretory Pathway (L16)
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unifying principle of vesicle trafficking involves what four things?
• cargo selection • vesicle budding from one compartment • transport • fusion with a second compartment
how can protein transport through the secretory pathway be visualized?
fluorescence microscopy of cells producing a GFP-membrane fusion protein
detection of compartment-sensitive oligosaccharide modifications?
transport of a membrane glycoprotein (VSVG) can be assayed by sensitivity to endoglycosidase D
cell-free assays for intercompartmental protein transport?
isolated golgi stacks from uninfected WT and infected mutant cell lines are combined in the test tube -> then VSVG is glycosylated • normally, VSVG is modified in the golgi by GlcNAc transferase • mutant cells do not perform this rxn
what three general ideas are necessary for cargo selection and budding?
• mechanical force: scission or propulsion/motility • target recognition: Rab GTPases • membrane fusion: SNAREs
what does coat assembly drive?
vesicle budding and selection of cargo
three major classes of vesicle coats
COPII COPI clathrin and adaptor proteins
what are the coats associated with secretory vesicles (TGN -> PM)?
unknown
two types of small GTPase associated w/ coating?
SAR1 and ARF - members of Ras GTPase superfamily
what are SAR1 and ARF associated with, respectively?
SAR1: COPII coats ARF: COPI and clathrin coats
when is SAR1 active?
when GEF exchanges a GDP for a GTP on the SAR1 protein
what makes SAR1 inactive?
when GAP exchanges a GTP for a GDP on the SAR1 protein
what is the function of Sec12 with SAR1?
activates it and recruits it to ER membrane - Sec12 is a GEF for SAR1
function of Sec23 for SAR1?
GAP - stimulates GTP hydrolysis after coat assembly
function of Sec24 for SAR1?
recruits cargo to the vesicle budding site
CFTR sorting signal
chloride channel that has a di-acidic sorting signal for packaging in COPII vesicles at the ER
mutation that causes cystic fibrosis
deltaF508 - blocks export of CFTR from the ER
function of Rab proteins
specify compartment identity
how do GTP-Rabs associate with membranes?
undergo a conformational change to expose a prenylated anchor that associates w/ membranes
name some Rab effector proteins?
• large protein complexes (exocyst) that mark specific sites • long, filamentous tethering proteins that anchor to acceptor • cytoskeletal motor proteins that facilitate targeting/ transport
function of SNAREs
• bring vesicle and target membranes into close apposition - may also confer specificity on targeting-cognate pairs of v- and t-SNARE
what happens to SNAREs once fused?
they are disassembled and recycled
functions of Golgi
• transport and packaging - modification and synthesis of oligosaccharides
describe Golgi
• has polarity: forming (cis) -> maturing (trans) cisternae • cis often near ER • gradations in staining