Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis.
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.
Sato
Ken
K
Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, PRESTO, Japan Science and Technology Agency, Hirosawa, Wako, Saitama. kensato@riken.jp
Nakano
Akihiko
A
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
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