Oxidative stress response mediated by the yeast Rho5 GTPase depends on the proper spatiotemporal distribution of its dimeric GEF

The small GTPase Rho5 acts as a central hub to mediate the yeast’s response to adverse environmental conditions, including oxidative stress, with the concomitant induction of mitophagy and apoptosis. A proper cellular stress response has been correlated with the rapid translocation of the GTPase to the mitochondria, which depends on its activating dimeric GDP/GTP exchange factor (GEF). Here, the small ALFA tag was attached to Rho5 or the GEF subunits Dck1 and Lmo1 to efficiently trap the functional fusion proteins to specific cellular membranes, i.e. the plasma membrane, the mitochon-drial outer membrane, or the nuclear membrane, via fusions of integral membrane proteins residing in these compartments with an ALFA nanobody. The trapped components were subjected to life-cell fluorescence microscopy in combination with GFP fusions of the GTPase or its GEF subunits to investigate their interaction in vivo . We found that the dimeric GEF tends to auto-assemble and form stable dimers independent of its intracellular localization. On the other hand, GFP-Rho5 does not stably colocalize with the trapped GEF, attributed to its transient interaction. Phenotypic analyses of strains with the misslocalized proteins indicate that for a proper oxidative stress response Lmo1 needs to associate with the plasma membrane. In contrast, Rho5 only exerts its role at the mitochondrial surface when it is there in its active conformation. These data underline the importance of the proper spatio-temporal distribution of Rho5-GTP during oxidative stress response.