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

Nature Structural & Molecular Biology volume 12, pages 167–174 (2005)Cite this article

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.

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Figure 1: FRET experimental design.
Figure 2: COPII vesicle formation from proteoliposomes.
Figure 3: FRET between reconstituted CFP-SNARE and YFP-Sec24/23p.
Figure 4: Sar1p-GTP hydrolysis and transient assembly of the prebudding complex as monitored by FRET.
Figure 5: Correlation between dissociation of prebudding complex and Sar1p-GTP hydrolysis on protein-free liposomes.
Figure 6: The presence of Sec12p prevents dissociation of prebudding complex.
Figure 7: Coat exchange on SNAREs during the Sar1p GTPase cycle.

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Acknowledgements

This work was supported by PRESTO, Japan Science and Technology Agency, by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan, and by a fund from the Bioarchitect Research Project of RIKEN.

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Authors and Affiliations

  1. Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, Saitama, 351-0198, Japan

    Ken Sato & Akihiko Nakano

  2. PRESTO, Japan Science and Technology Agency, Hirosawa, Wako, Saitama, 351-0198, Japan

    Ken Sato

  3. Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Akihiko Nakano

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  1. Ken Sato
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Correspondence to Ken Sato.

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Supplementary information

Supplementary Fig. 1

FRET between reconstituted MBP-CFP-Ufe1p and YFP-Sec24/23p. (PDF 414 kb)

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Sato, K., Nakano, A. Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis. Nat Struct Mol Biol 12, 167–174 (2005). https://doi.org/10.1038/nsmb893

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