Differential localization of two plant homologues of Rab5 GTPases in the secretory pathway

Abstract

Rab5 GTPases are key regulators of vesicular transport. Known plant Rab5 homologues ARA6 and RHA1 have been assigned to the endocytic and/or biosynthetic vesicular trafficking pathway, but conflicting reports in the literature justify further work on these two GTPases. In this project, binary vectors for Agrobacterium-mediated plant transformation were constructed to drive expression in tobacco leaf epidermis of GTP-trapped mutants of ARA6-green fluorescent protein (GFP) and Venus-RHA1. Confocal laser scanning microscopy revealed key differences in the subcellular localization of the two fluorescently tagged GTPase mutants. When present in the GTP-locked configuration ARA6-GFP is primarily found associated with the tonoplast, whereas Venus-RHA1 is significantly cytosolic. Co-expression of the two fluorescently tagged mutant GTPases with the Golgi markers ST-YFP and ST-CFP, respectively, suggest that neither ARA6 nor RHA1 mutants are mis-targeted to the Golgi apparatus and moreover, they do not show any significant colocalization with each other. The results are consistent with the notion that they differ in their roles within the endocytic and biosynthetic vesicular pathway. A future focus to continue this project would be to use GFP-tagged ARA7, the third plant Rab5 homologue, to verify if the high sequence homology between RHA1 and ARA7 warrants overlapping redundant functions or not.