NDLI: Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.

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Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.

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Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Chen, Y. T. Moore, H. P. Holcomb, C.
Description	Author Affiliation: Chen YT ( Department of Molecular and Cell Biology, University of California, Berkeley 94720.);
Abstract	Small GTP-binding proteins of the YPT/SEC4/Rab family have been shown to play an essential role in intracellular membrane trafficking. In mammals, Rab8 and Rab10 are the two small GTP-binding proteins identified so far that are closest to SEC4, an essential gene product involved in post-Golgi constitutive secretion in the yeast Saccharomyces cerevisiae. To study the localization of Rab proteins, we have expressed the cDNAs with an influenza virus hemagglutinin (HA) epitope tag at the N terminus. The feasibility of this method was tested by using yeast SEC4. HA-tagged SEC4 functionally complemented a temperature-sensitive sec4 mutant similarly to wild-type SEC4, indicating that the modified protein retained functional integrity. Monoclonal antibody 12CA5, raised against the HA tag, was used to determine the expression and localization of HA-tagged proteins after transfection. In stably transfected CHO and Swiss 3T3 cells, HA-tagged Rab8 was localized to the cell periphery, with the highest concentration in the ruffling areas. In contrast, epitope-tagged Rab10 expressed in CHO and BHK cells was concentrated on membranes in the perinuclear region. By light microscopy, the staining partially overlapped with that of a Golgi marker, beta-COP. Thus, despite the high degree homology of Rab8 and Rab10 (66% identity), the two proteins are localized to distinct cellular compartments. This approach should provide a general tool for the analyses of other members of the YPT/SEC4/rab gene family.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	14
Volume Number	90
Language	English
Publisher	National Academy of Sciences
Publisher Date	1993-08-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Cell Compartmentation GTP-Binding Proteins Isolation & Purification Rab GTP-Binding Proteins Animals Cells, Cultured Cloning, Molecular Epitopes Fluorescent Antibody Technique Biosynthesis Genetics Genetic Complementation Test Hemagglutinin Glycoproteins, Influenza Virus Hemagglutinins, Viral Molecular Sequence Data RNA, Messenger Recombinant Fusion Proteins Saccharomyces Cerevisiae Saccharomyces Cerevisiae Proteins Subcellular Fractions Transfection Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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