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

Abstract

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 Rho5 – Yet Another Player in Yeast Glucose Signaling

“…Another autophagy component, Atg21, was shown to interact directly with Rho5 (Singh et al, 2019). As Rho5 rapidly translocates to mitochondria under oxidative stress, it has been proposed to directly trigger mitophagy (Bischof et al, 2024b;Bischof et al, 2022). Whether or not this is related to its effect on Ras2 observed herein remains to be determined, given that Ras2 and PKA are also recruited to mitochondria by Hsp60 affecting their turnover (Portela and Rossi, 2020).…”

Functional Conservation of the Small GTPase Rho5/Rac1—A Tale of Yeast and Men

“…With regard to other factors influencing the intracellular distribution of Rho5, its translocation to mitochondria under oxidative stress requires the presence of both of its GEF subunits, whereas Dck1 and Lmo1 can be recruited independently of each other [ 38 , 120 ]. Apart from these findings and the need for its C-terminal modification, the exact mode of Rho5 recruitment to mitochondria remains elusive [ 30 , 120 ].…”

“…With regard to other factors influencing the intracellular distribution of Rho5, its translocation to mitochondria under oxidative stress requires the presence of both of its GEF subunits, whereas Dck1 and Lmo1 can be recruited independently of each other [ 38 , 120 ]. Apart from these findings and the need for its C-terminal modification, the exact mode of Rho5 recruitment to mitochondria remains elusive [ 30 , 120 ]. Evidence from studies on trapping Rho5 or its GEF subunits to different membranes indicates that in order to exert its function in oxidative stress response, the GTPase needs to be activated at the plasma membrane prior to its translocation to mitochondria.…”