Rab4 and Rab7 Define Distinct Nonoverlapping Endosomal Compartments*
- From the Department of Cell Biology, Utrecht University School of Medicine, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
- §Investigator of the Royal Netherlands Academy of Arts and Sciences. To whom correspondence should be addressed. Tel.: 31-302506474; Fax: -31-302541797; E-mail: pvander{at}knoware.nl .
Abstract
Several Rab GTPases have been localized to distinct compartments of the endocytic pathway. Rab4 is associated with early endosomes and recycling vesicles and regulates membrane recycling from early endosomes. Rab7 is localized to late endosomes and is involved in the regulation of membrane transport between late endosomes and lysosomes. Although Rab4 and Rab7 appear to regulate distinct transport events in endocytosis, it is not clear whether they perform their activities in related or entirely distinct intracellular compartments. To address this question, we generated stable cell lines expressing Rab4 tagged with a novel X31 influenza hemagglutinin (NH) epitope tag. These antibodies are characterized in this paper and were used to immunoisolate endocytic vesicles with cytoplasmically exposed NHRab4. Immunoisolated membranes contain internalized 125I-transferrin, but are devoid of Rab7. Confocal immunofluorescence microscopy showed that the early endosomal GTPases Rab4 and Rab5 both do not codistribute with Rab7 within the same cell. These observations suggest that each of the three Rab GTPases operationally defines a distinct station of the endocytic pathway.
Footnotes
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↵‡ Contributed equally to this work.
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↵* This work was supported by the Stichting de Drie Lichten and the Netherlands Organization for Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- TfR
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transferrin receptor
- hTfR
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human TfR
- Tf
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transferrin
- CHO
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Chinese hamster ovary
- GST
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glutathione S-transferase
- PBS
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phosphate-buffered saline.
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↵2P. van der Sluijs, E. Daro, D. Lewin, L. Gecez, A. Bachmann, P. Peters, and I. Mellman, submitted for publication.
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↵3Polymerase chain reaction primers used to generate the truncated NH epitope tag on Rab4 are available on request.
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- Received November 14, 1995.
- Revision received April 12, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
