In Vitro Formation of Recycling Vesicles from Endosomes Requires AP-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

Adriana Pagano, Pascal Crottet, Cristina Prescianotto-Baschong, and Martin Spiess*

Biozentrum, University of Basel, CH-4056 Basel, Switzerland

Monitoring Editor: Jean Gruenberg

The involvement of clathrinand associated adaptor proteins in receptor recycling from endosomesback to the plasma membrane is controversial. We have used anin vitro assay to identify the molecular requirements for theformation of recycling vesicles. Cells expressing the asialoglycoproteinreceptor H1, a typical recycling receptor, were surface-biotinylatedand then allowed to endocytose for 10 min. After stripping awaysurface-biotin, the cells were permeabilized and the cytosolwashed away. In a temperature-, cytosol-, and nucleotide-dependentmanner, the formation of sealed vesicles containing biotinylatedH1 could be reconstituted. Vesicle formation was strongly inhibitedupon immunodepletion of AP-1, but not of AP-2 or AP-3, fromthe cytosol, and was restored by readdition of purified AP-1.Vesicle formation was stimulated by supplemented clathrin, butinhibited by brefeldin A, consistent with the involvement ofARF1 and a brefeldin-sensitive guanine nucleotide exchange factor.The GTPase rab4, but not rab5, was required to generate endosome-derivedvesicles. Depletion of rabaptin-5/rabex-5, a known interactorof both rab4 and ${gamma}$ -adaptin, stimulated and addition of the purifiedprotein strongly inhibited vesicle production. The results indicatethat recycling is mediated by AP-1/clathrin coated vesiclesand regulated by rab4 and rabaptin-5/rabex-5.

^*Corresponding author. E-mail: Martin.Spiess{at}unibas.ch