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

Abstract

The small GTPase Rho5 of Saccharomyces cerevisiae is required for proper regulation of different signaling pathways, which includes the response to cell wall, osmotic, nutrient, and oxidative stress. We here show that proper in vivo function and intracellular distribution of Rho5 depends on its hypervariable 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 insertion only present in Rho5 of S. cerevisiae but not in its human homolog Rac1. Results from trapping GFP-Rho5 variants to the mitochondrial surface suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation to mitochondria in order to fulfil its role in oxidative stress response. These findings are supported by heterologous expression of a codon-optimized human RAC1 gene, which can only complement a yeast rho5 deletion in a chimeric fusion with RHO5 sequences that restore the correct spatiotemporal distribution of the encoded protein.

The Intracellular Distribution of the Small GTPase Rho5 and Its Dimeric Guanidine Nucleotide Exchange Factor Dck1/Lmo1 Determine Their Function in Oxidative Stress Response

“…This suggested that Rho5 at the plasma membrane suffices to trigger the appropriate response to oxidative stress under these conditions, i.e., that translocation of Rho5 and its GEF to mitochondria, is not required. In contrast to previous findings on the pronounced the hyper-sensitivity of strains with an activated RHO5 G12V allele towards oxidative stress [ 22 ], a pronounced difference as compared to the wild-type was found when the allele was fused with the MID2 coding sequence. Vice versa, a direct fusion of Rho5 with the transmembrane domain of Fis1, which confines the GTPase to the outer mitochondrial membrane, rendered the strains as hyper-resistant towards hydrogen peroxide as a rho5 deletion ( Figure 4 ).…”

“…Given their independent translocation, we wondered whether the GEF subunits activate Rho5 at the mitochondrial surface to trigger the cell’s response to oxidative stress. To investigate this hypothesis, Dck1 and Lmo1 were trapped to the mitochondria, as previously exercised with GFP-Rho5, by using a “GFP binder” [ 22 ]. Therefore, the genes encoding the GEF subunits were tagged with GFP at their native loci, and combined by crossing and tetrad analyses with a strain carrying a specific GFP nanobody fused to the transmembrane domain of the mitochondrial outer membrane Fis1 protein.…”

“…Studies on the domain structure of Rho5 showed an unusual extension of 98 amino acids in the C-terminal half, which precedes the polybasic region (PBR) and the CAAX-box common in Rho-type GTPases [ 21 ]. All three regions were required for proper stress-induced translocation of yeast Rho5 [ 22 ]. Moreover, the trapping of GFP-tagged Rho5 to the mitochondrial surface in vivo by a specific nanobody rendered it non-functional with regard to oxidative stress, with phenotypes similar to the rho5 deletion.…”

“…Moreover, the trapping of GFP-tagged Rho5 to the mitochondrial surface in vivo by a specific nanobody rendered it non-functional with regard to oxidative stress, with phenotypes similar to the rho5 deletion. It was thus proposed that the GTPase needs to be activated at the plasma membrane in order to fulfill its role in mitophagy and apoptosis [ 22 ].…”

Diverse Physiological Functions and Regulatory Mechanisms for Signal-Transducing Small GTPases

“…Takenaka et al [ 17 ] investigated the role of the Rho family small GTPase Rac1 in insulin-stimulated glucose uptake in adipocytes, demonstrating that Rac1 acts as a switch of insulin signaling downstream of the protein kinase Akt2. Sterk et al [ 18 ] analyzed the role of the small GTPase Rho5, the Saccharomyces cerevisiae ortholog of human Rac1, in oxidative stress response. In particular, they focused on the function of the hypervariable region at the carboxyterminal end, including a unique 98 amino acid insertion.…”