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Expression and Localization of Two Low Molecular Weight GTP-Binding Proteins, Rab8 and Rab10, by Epitope Tag
Yih-Tai Chen, Cherie Holcomb and Hsiao-Ping H. Moore
Proceedings of the National Academy of Sciences of the United States of America
Vol. 90, No. 14 (Jul. 15, 1993), pp. 6508-6512
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/2362535
Page Count: 5
You can always find the topics here!Topics: CHO cells, Proteins, Yeasts, Swiss 3T3 cells, Cell lines, Cells, Epitopes, Antibodies, Plant cells, Complementary DNA
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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, β-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.
Proceedings of the National Academy of Sciences of the United States of America © 1993 National Academy of Sciences