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

Ken Sato, Akihiko Nakano
2005 Nature Structural & Molecular Biology  
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
more » ... AREs 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. RESULTS CFP-SNARE proteoliposomes support vesicle budding FRET donor-acceptor pairs CFP and YFP were fused to the N termini of Bet1p/Sec22p and Sec24p, respectively. Recombinant CFP-Bet1p or CFP-Sec22p was expressed as Strep-tagged protein in Escherichia coli and purified in octylglucoside. His 6 -tagged YFP-Sec24p was overexpressed together with Sec23p in yeast and purified as a complex. Fusion with SNAREs or Sec24p did not affect the spectral properties of CFP and YFP (data not shown). These fusion proteins retained activity in COPII vesicle budding, as in vitro vesicle budding reactions using microsomes expressing either CFP-Bet1p or CFP-Sec22p supplied with purified COPII components including YFP-Sec24/23p (Fig. 1b) show that both CFP-Bet1p and CFP-Sec22p were packaged into COPII-coated vesicles with YFP-Sec24/23p. In contrast, an ER resident protein,
doi:10.1038/nsmb893 pmid:15665868 fatcat:lg65s2zdtrd6tea5axtjndc624