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

Rho5, the yeast homolog of human Rac1, is a small GTPase which regulates the cell response to nutrient and oxidative stress by inducing mitophagy and apoptosis. It is activated by a dimeric GEF composed of the subunits Dck1 and Lmo1. Upon stress, all three proteins rapidly translocate from the cell surface (Rho5) and a diffuse cytosolic distribution (Dck1 and Lmo1) to mitochondria, with translocation of the GTPase depending on both GEF subunits. We here show that the latter associate with mitochondria independent from each other and from Rho5. The trapping of Dck1-GFP or GFP-Lmo1 to the mitochondrial surface by a specific nanobody fused to the transmembrane domain (TMD) of Fis1 results in a loss of function, mimicking the phenotypes of the respective gene deletions, dck1 or lmo1. Direct fusion of Rho5 to Fis1TMD, i.e., permanent attachment to the mitochondria, also mimics the phenotypes of an rho5 deletion. Together, these data suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation in order to fulfill its function in the oxidative stress response. This notion is substantiated by the observation that strains carrying fusions of Rho5 to the cell wall integrity sensor Mid2, confining the GTPase to the plasma membrane, retained their function. We propose a model in which Rho5 activated at the plasma membrane represses the oxidative stress response under standard growth conditions. This repression is relieved upon its GEF-mediated translocation to mitochondria, thus triggering mitophagy and apoptosis.

The small GTPase KlRho5 responds to oxidative stress and affects cytokinesis.

Rho5 is the yeast homolog of the human small GTPase Rac1. We characterized the genes encoding Rho5 and the subunits of its dimeric activating guanine-nucleotide-exchange factor (GEF), Dck1 and Lmo1, in the yeast Kluyveromyces lactis. Rapid translocation of the three GFP-tagged components to mitochondria upon oxidative stress and carbon starvation indicate a similar function of KlRho5 in energy metabolism and mitochondrial dynamics as described for its Saccharomyces cerevisiae homolog. Accordingly, Klrho5 deletion mutants are hyper-resistant towards hydrogen peroxide. Moreover, synthetic lethalities of rho5 deletions with key components in nutrient sensing, such as sch9 and gpr1, are not conserved in K. lactis. Instead, Klrho5 deletion mutants display morphological defects with strengthened lateral cell walls and protruding bud scars. The latter result from aberrant cytokinesis, as observed by following the budding process in vivo and by transmission electron microscopy of the bud neck region. This phenotype can be suppressed by KlCDC42G12V, which encodes a hyper-active variant. Data from live-cell fluorescence microscopy support the notion that KlRho5 interferes with the actin moiety of the contractile actomyosin ring, with consequences different from those previously reported for mutants lacking myosin.

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

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 Small Yeast GTPase Rho5 and Its Dimeric GEF Dck1/Lmo1 Respond to Glucose Starvation.

Rho5 is a small GTPase of Saccharomyces cerevisiae and a homolog of mammalian Rac1. The latter regulates glucose metabolism and actin cytoskeleton dynamics, and its misregulation causes cancer and a variety of other diseases. In yeast, Rho5 has been implicated in different signal transduction pathways, governing cell wall integrity and the responses to high medium osmolarity and oxidative stress. It has also been proposed to affect mitophagy and apoptosis. Here, we demonstrate that Rho5 rapidly relocates from the plasma membrane to mitochondria upon glucose starvation, mediated by its dimeric GDP/GTP exchange factor (GEF) Dck1/Lmo1. A function in response to glucose availability is also suggested by synthetic genetic phenotypes of a rho5 deletion with gpr1, gpa2, and sch9 null mutants. On the other hand, the role of mammalian Rac1 in regulating the action cytoskeleton does not seem to be strongly conserved in S. cerevisiae Rho5. We propose that Rho5 serves as a central hub in integrating various stress conditions, including a crosstalk with the cAMP/PKA (cyclic AMP activating protein kinase A) and Sch9 branches of glucose signaling pathways.