In mammals, Rab5 and Rab7 play a specific and coordinated role in a sequential process during phagosome maturation. Here, we report that Rab5 and Rab7 in the enteric protozoan parasite Entamoeba histolytica, EhRab5 and EhRab7A, are involved in steps that are distinct from those known for mammals. EhRab5 and EhRab7A were localized to independent small vesicular structures at steady state. Priming with red blood cells induced the formation of large vacuoles associated with both EhRab5 and EhRab7A ("prephagosomal vacuoles (PPV)") in the amoeba within an incubation period of 5-10 min. PPV emerged de novo physically and distinct from phagosomes. PPV were gradually acidified and matured by fusion with lysosomes containing a digestive hydrolase, cysteine proteinase, and a membrane-permeabilizing peptide amoebapore. After EhRab5 dissociated from PPV, 5-10 min later, the EhRab7A-PPV fused with phagosomes, and EhRab7A finally dissociated from the phagosomes. Immunoelectron and light micrographs showed that PPV contained small vesicle-like structures containing fluid-phase markers and amoebapores, which were not evenly distributed within PPV, suggesting that the mechanism was similar to multivesicular body formation in PPV generation. In contrast to Rab5 from other organisms, EhRab5 was involved exclusively in phagocytosis, but not in endocytosis. Overexpression of wild-type EhRab5 enhanced phagocytosis and the transport of amoebapore to phagosomes. Conversely, expression of an EhRab5Q67L GTP form mutant impaired the formation of PPV and phagocytosis. Altogether, we propose that the amoebic Rab5 plays an important role in the formation of unique vacuoles, which is essential for engulfment of erythrocytes and important for packaging of lysosomal hydrolases, prior to the targeting to phagosomes.

Vesicular trafficking plays an important role in a virulence mechanism of the enteric protozoan parasite Entamoeba histolytica as secreted and lysosomal cysteine protease (CP) contributes to both cytolysis of tissues and degradation of internalized host cells. Despite the primary importance of intracellular sorting in pathogenesis, the molecular mechanism of CP trafficking remains largely unknown. In this report we demonstrate that transport of CP is regulated through a specific interaction of Rab7A small GTPase (EhRab7A) with the retromerlike complex. The amoebic retromerlike complex composed of Vps26, Vps29, and Vps35 was identified as EhRab7A-binding proteins. The amoebic retromerlike complex specifically bound to GTP-EhRab7A, but not GDP-EhRab7A through the direct binding via the carboxy terminus of EhVps26. In erythrophagocytosis the retromerlike complex was recruited to prephagosomal vacuoles, the unique preparatory vacuole of digestive enzymes, and later to phagosomes. This dynamism was indistinguishable from that of EhRab7A, and consistent with the premise that the retromerlike complex is involved in the retrograde transport of putative hydrolase receptor(s) from preparatory vacuoles and phagosomes to the Golgi apparatus. EhRab7A overexpression caused enlargement of lysosomes and decrease of the cellular CP activity. The reduced CP activity was restored by the coexpression of EhVps26, implying that the EhRab7A-mediated transport of CP to phagosomes is regulated by the retromerlike complex. INTRODUCTIONRab GTPases play an essential role to regulate intracellular membrane trafficking. The compartmentalization and functions of Rab proteins are modulated at multiple layers of mechanisms including isoprenylation of the carboxy terminus, nucleotide exchange, and binding of specific effector molecules (Stenmark and Olkkonen, 2001;Takai et al., 2001;Tuvim et al., 2001). Among these modulators, effectors play key roles in the control of 7-90 Rab proteins depending on organisms from fission yeast and amoeba to mammals and plants. A variety of effectors have been identified and shown to interact with specific Rab protein. For instance, regulatory secretions of neurotransmitters from neurons or insulin from pancreatic ␤-cells are regulated by the specific interaction of Rab3 with its effectors (Jahn and Sudhof, 1999). At least four proteins, Rabphilin3, Rim1, Rim2, and Noc2, are known to bind Rab3 and recruit cAMP-GEFII, 14 -3-3, and zyxin, respectively, to regulate cAMP responsiveness, phosphoserine-dependent signaling, and the interaction with cytoskeleton (Kotake et al., 1997;Ozaki et al., 2000;Sun et al., 2003). The specificity of the Rab-effector interaction is attributable to primary sequence motifs in only a few cases including exophilins or Slip/Slac2, Rab3, and Rab27 effectors (Izumi et al., 2003). However, the majority of Rab effectors lack recognizable conserved binding motifs. For instance, Rab5 effectors, rabaptin-5, Rabex-5, EEA1, Rabenosyn-5, hVps34/p150, p110␤/p35␣, rabip4Ј, and rabankyrin-5, which a...

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SummaryRab7 small GTPase plays a crucial role in the regulation of trafficking to late endosomes, lysosomes and phagosomes. While most eukaryotes encode a single Rab7, the parasitic protist Entamoeba histolytica possesses nine Rab7. In this study, to understand the significance of the presence of multiple Rab7 isotypes, a role of two representative Rab7 isotypes, EhRab7A and EhRab7B, was investigated. EhRab7B was exclusively localized to acidic vacuoles containing 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. Overexpression 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 acidification and missecretion of lysosomal proteins, while EhRab7A-GTP enhanced acidification but did not affect either intracellular or secreted cysteine protease activity. Expression of either EhRab7B or EhRab7B-GTP mutant caused defect in phagocytosis, concomitant with the disturbed formation and disassembly of prephagosomal vacuoles, the compartment previously shown to be linked to efficient ingestion.Altogether, these data indicate that the two Rab7 isotypes play distinct but co-ordinated roles in lysosome and phagosome biogenesis.