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

• Published in: Proceedings of the National Academy of Sciences - PNAS 1993-07, Vol.90 (14), p.6508-6512
• Main Authors: Chen, Y.T, Holcomb, C, Moore, H.P.H
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Antibodies; ANTICORPS MONOCLONAL; ANTICUERPOS MONOCLONALES; Base Sequence; Binding and carrier proteins; Biological and medical sciences; CARCINOGENOS; Cell Compartmentation; Cell lines; Cells; Cells, Cultured; Cellular biology; CHO cells; Cloning, Molecular; Complementary DNA; Epitopes; EXPRESION GENICA; EXPRESSION DES GENES; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; GENE; GENES; Genetic Complementation Test; Genetics; GTP-Binding Proteins - biosynthesis; GTP-Binding Proteins - genetics; GTP-Binding Proteins - isolation & purification; Hemagglutinin Glycoproteins, Influenza Virus; Hemagglutinins, Viral - biosynthesis; Hemagglutinins, Viral - genetics; Hemagglutinins, Viral - isolation & purification; Humans; LEVADURA; LEVURE; Mammalia; Membranes; Molecular Sequence Data; NEOPLASMAS; NEOPLASME; PROTEINAS; PROTEINE; Proteins; rab GTP-Binding Proteins; Rats; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; RNA, Messenger - genetics; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Subcellular Fractions; SUBSTANCE CANCERIGENE; Swiss 3T3 cells; Transfection; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.14.6508

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 family