Two Rab7 isotypes, EhRab7A and EhRab7B, play distinct roles in biogenesis of lysosomes and phagosomes in the enteric protozoan parasite Entamoeba histolytica Yumiko Saito-Nakano, 1 Biswa Nath Mitra, 2 Kumiko Nakada-Tsukui, 2 Dan Sato 2 and Tomoyoshi Nozaki 2 * 1 Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. 2 Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Summary Rab7 small GTPase plays a crucial role in the regula- tion of trafficking to late endosomes, lysosomes and phagosomes. While most eukaryotes encode a single Rab7, the parasitic protist Entamoeba histolytica pos- sesses nine Rab7. In this study, to understand the significance of the presence of multiple Rab7 iso- types, a role of two representative Rab7 isotypes, EhRab7A and EhRab7B, was investigated. EhRab7B was exclusively localized to acidic vacuoles contain- ing lysosomal proteins, e.g. amoebapore-A and cysteine protease. This lysosome localization of EhRab7B was in good contrast to EhRab7A, localized to a non-acidic compartment in steady state, and only partially colocalized with lysosomal proteins. Overex- pression of EhRab7B resulted in augmentation of late endosome/lysosome acidification, similar to the EhRab7A overexpression. Expression of EhRab7B- GTP mutant caused dominant-negative phenotypes including decrease in late endosome/lysosome acidi- fication and missecretion of lysosomal proteins, while EhRab7A-GTP enhanced acidification but did not affect either intracellular or secreted cysteine pro- tease activity. Expression of either EhRab7B or EhRab7B-GTP mutant caused defect in phagocytosis, concomitant with the disturbed formation and disas- sembly of prephagosomal vacuoles, the compartment previously shown to be linked to efficient ingestion. Altogether, these data indicate that the two Rab7 iso- types play distinct but co-ordinated roles in lysosome and phagosome biogenesis. Introduction Lysosomes serve as a compartment to degrade endocy- tosed materials with various hydrolytic and degradative proteins. Lysosomes also play a role as a compartment to process, activate and store proteins secreted extracellu- larly (Mullins and Bonifacino, 2001; Bowers and Stevens, 2005). Among a number of molecules involved in the lysosome biogenesis, one of the most important players is Rab7 small GTPase (Mullins and Bonifacino, 2001). Roles of Rab7 are significantly divergent between organ- isms and cell types; Rab7 plays a role on several distinct steps of endosomal or lysosomal trafficking (Feng et al., 1995; Meresse et al., 1995; Vitelli et al., 1997; Bucci et al., 2000). In baby hamster kidney cells, Rab7 was shown to mediate the early-to-late endosomal transport (Feng et al., 1995; Vitelli et al., 1997), whereas in HeLa cells, Rab7 was mainly associated with lysosomes and involved in lysosome acidification and transport of lysosomal pro- teins, but not of late endosomal proteins (Meresse et al., 1995; Bucci et al., 2000). Moreover, Rab7 was also shown to be required for fusion of late endosomes and lysosomes with primary phagosomes in professional phagocytes (Vieira et al., 2003). In yeast, Ypt7p, the yeast Rab7 homologue, was also shown to be involved in trans- port from the late endosomes to the central vacuole, the yeast lysosome equivalent, and to be required for homo- typic fusion of the vacuoles (Wichmann et al., 1992; Schimmoller and Riezman, 1993; Haas et al., 1995). While many uni- and multicellular eukaryotes including Saccharomyces cerevisiae, Trypanosoma brucei, Plas- modium falciparum, Caenorhabditis elegans, Drosophila melanogaster, human, encode a single Rab7 (Pereira- Leal and Seabra, 2001; Quevillon et al., 2003; Ackers et al., 2005; Berriman et al., 2005), some protozoan para- sites including Trichomonas vaginalis and Entamoeba his- tolytica have three to nine Rab7 isotypes (Lal et al., 2005; Saito-Nakano et al., 2005), which is similar to plants such as Lotus japonicus and Arabidopsis thaliana, having four Received 9 November, 2006; revised 24 January, 2007; accepted 30 January, 2007. *For correspondence. E-mail nozaki@med.gunma- u.ac.jp; Tel. (+81) 27 2208020; Fax (+81) 27 2208025. Cellular Microbiology (2007) 9(7), 1796–1808 doi:10.1111/j.1462-5822.2007.00915.x First published online 13 March 2007 © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd