Identification of Dck1 and Lmo1 as upstream regulators of the small GTPase Rho5 in Saccharomyces cerevisiae

Abstract

SummaryThe exact function and regulation of the small GTPase Rho5, a putative homolog of mammalian Rac1, in the yeast Saccharomyces cerevisiae have not yet been elucidated. In a genetic screen initially designed to identify novel regulators of cell wall integrity signaling, we identified the homologs of mammalian DOCK1 (Dck1) and ELMO (Lmo1) as upstream components which regulate Rho5. Deletion mutants in any of the encoding genes (DCK1, LMO1, RHO5) showed hyperresistance to cell wall stress agents, demonstrating a function in cell wall integrity signaling. Live-cell fluorescence microscopy showed that Dck1, Lmo1 and Rho5 quickly relocate to mitochondria under oxidative stress and cell viability assays indicate a role of Dck1/ Lmo1/Rho5 signaling in triggering cell death as a response to hydrogen peroxide treatment. A regulatory role in autophagy/mitophagy is suggested by the colocalization of Rho5 with autophagic markers and the decreased mitochondrial turnover observed in dck1, lmo1 and rho5 deletion mutants. Rho5 activation may thus serve as a central hub for the integration of different signaling pathways.

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

“…This is in contrast to Dck1 and Lmo1 which show a diffuse cytoplasmic distribution in the absence of oxidative stress, with the occasional formation of foci in which the two proteins colocalize (Figure 1B and [10]). We wondered whether these foci are stable and investigated a relation to certain growth conditions, with a focus on glucose availability.…”

“…We previously found that Rho5 is associated with the plasma membrane under normal growth conditions and rapidly switches to mitochondria under oxidative stress, the latter being dependent on its dimeric GEF Dck1/Lmo1 [10]. This is in contrast to Dck1 and Lmo1 which show a diffuse cytoplasmic distribution in the absence of oxidative stress, with the occasional formation of foci in which the two proteins colocalize (Figure 1B and [10]).…”

“…Confirming our previous data, the foci only formed in the presence of Lmo1, reflecting the interdependence of the two GEF subunits. We previously showed that they do not colocalize with any known organelles or vesicles [10], and therefore speculate that the foci form as a transient Dck1/Lmo1 complex in response to subtle changes in intracellular conditions, which ultimately serve to transport active Rho5 to mitochondria.…”

“…Therefore, epistasis analyses were performed by crossing rho5 deletion strains with different mutants impaired in glucose signaling. In view of the previously detected relation of Rho5 to autophagy and mitophagy [9,10], and the reported synthetic lethality of gpr1 and gpa2 with sch9 (; accessed on 20 June 2018), we also included the latter deletion (see Figure 1A for proposed interactions). Isogenic diploid strains being heterozygous for both rho5 and either of these deletions were subjected to tetrad analyses.…”

“…We recently showed that intracellular distribution of Rho5 is driven by association with its dimeric GDP/GTP exchange factor (GEF) Dck1/Lmo1 under conditions of oxidative stress. Thus, upon addition of hydrogen peroxide the trimeric complex rapidly relocates from a diffuse and partially patchy cytosolic distribution (Dck1/Lmo1; compare Figure 1B) and the plasma membrane (Rho5) to mitochondria, providing a mechanical link to the roles observed in mitophagy and apoptosis [10]. Interestingly, deletion of RHO5 , and also of DCK1 or LMO1 , led to hyper-resistance towards the cell wall stress agents Calcofluor white and Congo red, supporting the originally proposed link to CWI signaling [4,10].…”