Reconstitution of Rab4-dependent vesicle formation in vitro

Pagano, Adriana and Spiess, Martin. (2005) Reconstitution of Rab4-dependent vesicle formation in vitro. Methods in enzymology, vol. 403. pp. 81-92.

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Official URL: http://edoc.unibas.ch/dok/A5258039

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We have developed an in vitro assay to reconstitute the formation of endosomal recycling vesicles. To achieve specificity for endosomes as the donor organelle, cells are surface-biotinylated and allowed to endocytose for 10 min, after which the remaining surface-biotin is stripped off. The cells are then permeabilized and the cytosol washed away. Upon addition of exogenous cytosol and energy, sealed vesicles containing biotinylated recycling receptors are produced. Modification of the cytosol, for example, by immunodepletion or addition of purified proteins, allows the identification of proteins involved in vesicle formation. The results show that recycling is mediated by AP-1/clathrin-coated vesicles, requires Rab4, and is negatively regulated by rabaptin-5/rabex-5.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Spiess)
UniBasel Contributors:Spiess, Martin
Item Type:Article, refereed
Article Subtype:Research Article
Note:Also published in: GTPases regulating membrane targeting and fusion. - Amsterdam : Elsevier, 2005. - S. 81-92 -- Publication type according to Uni Basel Research Database: Journal article
Last Modified:07 Aug 2015 12:05
Deposited On:22 Mar 2012 13:59

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