A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance.All eukaryotic cells are compartmentalized into distinct membrane-bound organelles and require tightly regulated transport of proteins and lipids between these compartments. Members of the Rab family of small GTPases are major regulators of protein and lipid traffic in the secretory and endocytic pathways (46,65). The action of Rab proteins in membrane transport was discovered with the identification of the SEC4 and YPT1 genes in yeast (Saccharomyces cerevisiae) (51, 52) and related proteins, termed Rab proteins, from mammals (10). Rab proteins comprise the most numerous subfamily of the Ras superfamily, and many are functionally uncharacterized. It is not clear whether Rab proteins regulate events that take place in the donor compartment, the vesicle or transport carrier, the acceptor compartment, or multiple locations. Also not known is whether a function(s) can be described for Rab proteins in general or whether this must be considered on a case-by-case basis. However, it is clear that Rab proteins are essential for eukaryotic cells and absolutely required for the function of all organelles connected by SNARE-mediated membrane traffic.With the completion of eukaryotic genome sequencing projects, there have been efforts to catalog the numbers of GTPase superfamily proteins. Membrane traffic in higher eukaryotes connects multiple compartments, and one reflection of this complexity may be the finding that humans have at least 60 different Rab family members (24,55,57). Other eukaryotes have fewer Rab proteins; Caenorhabditis elegans has 29 family members, Drosophila melanogaster has 26 members, and the yeast Saccharomyces cerevisiae has 11 members. As a model system, the 11 Rab proteins of the single-celled eukaryotic microbe S. cerevisiae can be considered the most minimal "membrome" (24), as other single-celle...

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