The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae. Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant. Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place. The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes. Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe. The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study.
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March 2003
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Research Article|
March 01 2003
In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles
Lorena ARRASTUA;
Lorena ARRASTUA
∗Faculty of Chemistry, Biochemistry II, University of the Basque Country, P.O. Box 1072, E-20080 San Sebastián, Spain,
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Eider SAN SEBASTIAN;
Eider SAN SEBASTIAN
∗Faculty of Chemistry, Biochemistry II, University of the Basque Country, P.O. Box 1072, E-20080 San Sebastián, Spain,
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Ana F. QUINCOCES;
Ana F. QUINCOCES
∗Faculty of Chemistry, Biochemistry II, University of the Basque Country, P.O. Box 1072, E-20080 San Sebastián, Spain,
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Claude ANTONY;
Claude ANTONY
†Institute Curie, Centre National de la Recherche Scientifique UMR 144, Laboratoire Mécanismes Moléculaires du Transport Intracellulaire, F-75248 Paris Cedex, France
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Unai UGALDE
Unai UGALDE
1
∗Faculty of Chemistry, Biochemistry II, University of the Basque Country, P.O. Box 1072, E-20080 San Sebastián, Spain,
1To whom correspondence should be addressed (e-mail qppugmau@sc.ehu.es).
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Publisher: Portland Press Ltd
Received:
November 06 2002
Accepted:
November 15 2002
Accepted Manuscript online:
November 15 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 370 (2): 641–649.
Article history
Received:
November 06 2002
Accepted:
November 15 2002
Accepted Manuscript online:
November 15 2002
Citation
Lorena ARRASTUA, Eider SAN SEBASTIAN, Ana F. QUINCOCES, Claude ANTONY, Unai UGALDE; In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles. Biochem J 1 March 2003; 370 (2): 641–649. doi: https://doi.org/10.1042/bj20021736
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