In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5.
| Title: | In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5. |
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| Authors: | Pagano A; Biozentrum, University of Basel, CH-4056 Basel, Switzerland.; Crottet P; Prescianotto-Baschong C; Spiess M |
| Source: | Molecular biology of the cell [Mol Biol Cell] 2004 Nov; Vol. 15 (11), pp. 4990-5000. Date of Electronic Publication: 2004 Aug 25. |
| Publication Type: | Journal Article; Research Support, Non-U.S. Gov't |
| Language: | English |
| Journal Info: | Publisher: American Society for Cell Biology Country of Publication: United States NLM ID: 9201390 Publication Model: Print-Electronic Cited Medium: Print ISSN: 1059-1524 (Print) Linking ISSN: 10591524 NLM ISO Abbreviation: Mol Biol Cell Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Bethesda, MD : American Society for Cell Biology, c1992- |
| MeSH Terms: | Endosomes/*physiology ; Transcription Factor AP-1/*physiology ; Vesicular Transport Proteins/*physiology ; rab4 GTP-Binding Proteins/*physiology; Adaptor Protein Complex 2/metabolism ; Adaptor Protein Complex 3/metabolism ; Biotin/chemistry ; Cell Membrane/metabolism ; Cytosol/metabolism ; Histones/metabolism ; Transcription Factor AP-1/metabolism ; Animals ; Biotinylation ; Cattle ; Microscopy, Electron ; Models, Biological ; Protein Binding ; Temperature ; Time Factors |
| Abstract: | The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5. |
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| Substance Nomenclature: | 0 (Adaptor Protein Complex 2); 0 (Adaptor Protein Complex 3); 0 (Histones); 0 (RABEP1 protein, human); 0 (Transcription Factor AP-1); 0 (Vesicular Transport Proteins); 6SO6U10H04 (Biotin); EC 3.6.5.2 (rab4 GTP-Binding Proteins) |
| Entry Date(s): | Date Created: 20040828 Date Completed: 20050408 Latest Revision: 20241119 |
| Update Code: | 20260130 |
| PubMed Central ID: | PMC524758 |
| DOI: | 10.1091/mbc.e04-04-0355 |
| PMID: | 15331762 |
| Database: | MEDLINE |
Journal Article; Research Support, Non-U.S. Gov't