Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis

Ken Sato; Akihiko Nakano

doi:10.1038/nsmb893

Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis

Nat Struct Mol Biol. 2005 Feb;12(2):167-74. doi: 10.1038/nsmb893. Epub 2005 Jan 23.

Authors

Ken Sato¹, Akihiko Nakano

Affiliation

¹ Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, PRESTO, Japan Science and Technology Agency, Hirosawa, Wako, Saitama. kensato@riken.jp

PMID: 15665868
DOI: 10.1038/nsmb893

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

COP-Coated Vesicles / metabolism*
Fluorescence Resonance Energy Transfer
GTP Phosphohydrolases / metabolism
GTPase-Activating Proteins
Guanine Nucleotide Exchange Factors
Guanosine Diphosphate / metabolism
Guanosine Triphosphate / metabolism*
Hydrolysis
Kinetics
Liposomes / metabolism
Membrane Glycoproteins / metabolism
Membrane Proteins / metabolism
Monomeric GTP-Binding Proteins / metabolism*
Protein Subunits / metabolism
Saccharomyces cerevisiae / genetics
Saccharomyces cerevisiae / metabolism
Saccharomyces cerevisiae Proteins / metabolism*
Time Factors
Vesicular Transport Proteins

Substances

GTPase-Activating Proteins
Guanine Nucleotide Exchange Factors
Liposomes
Membrane Glycoproteins
Membrane Proteins
Protein Subunits
SEC12 protein, S cerevisiae
SEC23 protein, S cerevisiae
SEC24 protein, S cerevisiae
Saccharomyces cerevisiae Proteins
Vesicular Transport Proteins
Guanosine Diphosphate
Guanosine Triphosphate
GTP Phosphohydrolases
Monomeric GTP-Binding Proteins
SAR1 protein, S cerevisiae