title: Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis.
abstract: COPII coat proteins are required for direct capture of cargo and SNARE proteins into transport vesicles from the endoplasmic reticulum (ER). Cargo and SNARE capture occurs during the formation of a 'prebudding complex' comprising a cargo, Sar1p-GTP and the COPII subunits Sec23/24p. The assembly and disassembly cycle of the prebudding complex on ER membranes is coupled to the Sar1p GTPase cycle. Using FRET to monitor a single round of Sec23/24p binding and dissociation from SNAREs in reconstituted liposomes, we show that Sec23/24p dissociates from v-SNARE and complexed t-SNARE with kinetics slower than Sar1p-GTP hydrolysis. Once Sec23/24p becomes associated with v-SNARE or complexed t-SNARE, the complex remains assembled during multiple rounds of Sar1p-GTP hydrolysis mediated by the GDP-GTP exchange factor Sec12p. These data suggest a model for the maintenance of kinetically stable prebudding complexes during the Sar1p GTPase cycle that regulates cargo sorting into transport vesicles.
authors.affiliation: Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, PRESTO, Japan Science and Technology Agency, Hirosawa, Wako, Saitama. kensato@riken.jp
authors.forename: Ken
authors.initials: K
authors.lastname: Sato
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakano
mesh_terms: D022181:COP-Coated Vesicles / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013997:Time Factors; D033921:Vesicular Transport Proteins
chemical_list: D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008081:Liposomes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D021122:Protein Subunits; C071239:SEC12 protein, S cerevisiae; C079037:SEC23 protein, S cerevisiae; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae