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

摘要: 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 two small identified so far that closest SEC4, gene product involved post-Golgi constitutive secretion yeast Saccharomyces cerevisiae. To study localization Rab proteins, we expressed cDNAs with influenza virus hemagglutinin (HA) epitope tag at N terminus. The feasibility this method was tested by using SEC4. HA-tagged SEC4 functionally complemented a temperature-sensitive sec4 mutant similarly wild-type indicating modified protein retained functional integrity. Monoclonal antibody 12CA5, raised against HA tag, used determine expression after transfection. stably transfected CHO Swiss 3T3 cells, localized cell periphery, highest concentration ruffling areas. contrast, epitope-tagged BHK cells concentrated on membranes perinuclear region. By light microscopy, staining partially overlapped Golgi marker, beta-COP. Thus, despite high degree homology (66% identity), distinct cellular compartments. This approach should provide general tool for analyses other members YPT/SEC4/rab family.