Analysis of Functional Domains in Rho5, the Yeast Homolog of Human Rac1 GTPase, in Oxidative Stress Response

The small GTPase Rho5 ofSaccharomyces cerevisiaeis required for proper regulation ofdifferent signaling pathways, which includes the response to cell wall, osmotic, nutrient, and oxidativestress. We here show that properin vivofunction and intracellular distribution of Rho5 depends on itshypervariable region at the carboxyterminal end, which includes the CAAX box for lipid modification,a preceding polybasic region (PBR) carrying a serine residue, and a 98 amino acid–specific insertiononly present in Rho5 ofS. cerevisiaebut not in its human homolog Rac1. Results from trappingGFP-Rho5 variants to the mitochondrial surface suggest that the GTPase needs to be activated atthe plasma membrane prior to its translocation to mitochondria in order to fulfil its role in oxidativestress response. These findings are supported by heterologous expression of a codon-optimizedhumanRAC1gene, which can only complement a yeastrho5deletion in a chimeric fusion withRHO5sequences that restore the correct spatiotemporal distribution of the encoded protein