How can mammalian Rab small GTPases be comprehensively analyzed?: Development of new tools to comprehensively analyze mammalian Rabs in membrane traffic

Abstract

Small GTPase Rabs constitute the largest family of membrane trafficking proteins that are conserved in all eukaryotic cells. The number of different Rab isoforms in multicellular organisms is usually greater than that in unicellular organisms (e.g., approximately 60 different Rabs in each species of mammals investigated versus approximately 10 Rabs in yeasts). The expansion of Rab isoforms in mammals is often regarded as due to the acquisition of specialized membrane trafficking events in the specialized cell types of higher eukaryotes. However, because of their large numbers the precise function of most mammalian Rab isoforms is still unknown. The recent development of new tools for comprehensive analysis (e.g., Rab panels) has paved the way for systematic investigation of the involvement of mammalian Rab isoforms in specialized membrane trafficking events. The tools include collections of enhanced green fluorescent protein (EGFP)-tagged mouse and human Rabs, FLAG-tagged Rabs, glutathione S-transferase (GST)-tagged Rabs, Gal4-binding domain (GBD)-tagged Rabs, Tre-2/Bub2/Cdc16 (TBC) domain-containing Rab-GTPase-activating proteins (GAPs), and small interfering RNAs. EGFP-Rabs are used to screen for Rabs that are localized on specific organelles and regulate their transport, and GST-Rabs and GBD-Rabs are used to screen for novel Rab effectors by GST pull-down assays and yeast two-hybrid assays, respectively. Combined use of these tools now makes it possible to efficiently determine the function of mammalian Rab isoforms in membrane traffic. This article reviews the development of new tools for systematic analysis of Rab proteins and their application to Rab-mediated membrane traffic.