Abstract
Rab5 is a key regulator of early endocytosis by promoting early endosomal fusion and motility. In this study, we have unexpectedly found distinct properties of the two Rab5 homologs (MoRab5A and MoRab5B) from Magnaporthe oryzae, a pathogenic fungus in plants whose infection causes rice blast disease. Like mammalian Rab5, MoRab5A and MoRab5B can bind to several Rab5 effectors in a GTP-dependent manner, including EEA1, Rabenosyn-5, and Rabaptin-5. However, MoRab5A shows distinct binding characteristics in the sense that both the wild-type and the GTP hydrolysis-defective constitutively active mutant bind the effectors equally well in GST pull-down assays, suggesting that MoRab5A is defective in GTP hydrolysis and mostly in the GTP-bound conformation in the cell. Indeed, GTP hydrolysis assays indicate that MoRab5A GTPase activity is dramatically lower than MoRab5B and human Rab5 and is insensitive to RabGAP5 stimulation. We have further identified a Pro residue in the switch I region largely responsible for the distinct MoRab5A properties by characterization of MoRab5A and MoRab5B chimeras and mutagenesis. The differences between MoRab5A and MoRab5B extend to their functions in the cell. Although they both target to early endosomes, only MoRab5B closely resembles human Rab5 in promoting early endosome fusion and stimulating fluid phase endocytosis. In contrast, MoRab5A correlates with another related early endosomal Rab, Rab22, in terms of the presence of the switch I Pro residue and the blocked GTPase activity. Our data thus identify MoRab5B as the Rab5 ortholog and suggest that MoRab5A specializes to perform a non-redundant function in endosomal sorting.
Highlights
It is unclear if the two Rab5 homologs in M. oryzae (MoRab5A and MoRab5B) are functional Rab5 orthologs
Rab5 normally targets to early endosomes and promotes early endosome fusion and endocytosis (16 –20). We find that both MoRab5A and MoRab5B localize to early endosomes but only MoRab5B potently stimulates endocytosis in mammalian cells like human Rab5
To determine if MoRab5A or MoRab5B may represent a functional ortholog of mammalian Rab5, we compared the biochemical properties of MoRab5A and MoRab5B with human Rab5 (hRab5) and investigated if MoRab5A and MoRab5B can mimic hRab5 functions in mammalian cells
Summary
It is unclear if the two Rab homologs in M. oryzae (MoRab5A and MoRab5B) are functional Rab orthologs. Phylogenetic information alone cannot rule out the possibility that one of the currently annotated Rab homologs in lower eukaryotes such as fungi may represent a functional counterpart for one of the early endosomal Rab subfamily members rather than Rab itself in mammalian cells To address this issue, we have cloned the two Rab homologs (MGG_06241 and MGG_01185 termed here as MoRab5A and MoRab5B) from Magnaporthe oryzae (M. orzyae), which is a pathogenic fungus in plants and causes rice blast disease [15], and characterized their biochemical properties and determined if they exhibit the same function in stimulating endocytosis in. It is well documented that constitutively active (CA) GTPase-defective mutants of Rab (e.g. the Gln to Leu mutation in hRab5) [19, 20] dramatically increase the size of early endosomes in cells by promoting endosome fusion In this regard, MoRab5B closely resembles hRab, but not MoRab5A. Taken together with their interaction profiles with a panel of effectors including Rabaptin-5 [21], Rabenosyn-5 [22], and EEA1 [23], we conclude that MoRab5B represents the authentic ortholog of mammalian Rab while MoRab5A plays a distinct specialized function, possibly in endosomal sorting
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