Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.
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.
Chen
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YT
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Holcomb
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Moore
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HP
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D005816:Genetic Complementation Test; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013347:Subcellular Fractions; D014162:Transfection; D020691:rab GTP-Binding Proteins
D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D012333:RNA, Messenger; C082378:Rab10 protein, rat; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; C082377:RAB8A protein, human; C513177:Rab8b protein, rat; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins