Genome-Wide Studies of Rho5-Interacting Proteins That Are Involved in Oxidant-Induced Cell Death in Budding Yeast

Abstract

Rho GTPases play critical roles in cell proliferation and cell death in many species. As in animal cells, cells of the budding yeast Saccharomyces cerevisiae undergo regulated cell death under various physiological conditions and upon exposure to external stress. The Rho5 GTPase is necessary for oxidant-induced cell death, and cells expressing a constitutively active GTP-locked Rho5 are hypersensitive to oxidants. Yet how Rho5 regulates yeast cell death has been poorly understood. To identify genes that are involved in the Rho5 -mediated cell death program, we performed two complementary genome-wide screens: one screen for oxidant-resistant deletion mutants and another screen for Rho5 -associated proteins. Functional enrichment and interaction network analysis revealed enrichment for genes in pathways related to metabolism, transport, and plasma membrane organization. In particular, we find that ATG21 , which is known to be involved in the CVT (Cytoplasm-to-Vacuole Targeting) pathway and mitophagy, is necessary for cell death induced by oxidants. Cells lacking Atg21 exhibit little cell death upon exposure to oxidants even when the GTP-locked Rho5 is expressed. Moreover, Atg21 interacts with Rho5 preferentially in its GTP-bound state, suggesting that Atg21 is a downstream target of Rho5 in oxidant-induced cell death. Given the high degree of conservation of Rho GTPases and autophagy from yeast to human, this study may provide insight into regulated cell death in eukaryotes in general.

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

“…Supporting this notion is the hydrophilic, probably surface-exposed nature of this sequence predicted by secondary structure analyses. In a recent paper, Singh and co-workers reported on a large number of putative Rho5 interactors, comprising, amongst others, the two eisosome components Lsp1 and Sur7 [26]. Lsp1 directs the membrane invaginations typical for eisosomes through its BAR domain [27].…”

“…Pkc1 is a key component of the CWI pathway, to which Rho5 function was found to be related [9]. An evolutionary conserved interaction between Rho5 and Pkc1 would thus be an option, although it was neither detected in the work of Singh and co-workers [26] nor in direct two-hybrid interaction assays with the HR1 domains of Pkc1 [31]. Yet, since both assays were performed under standard growth conditions, it cannot be ruled out that an interaction might still occur in specific stress situations.…”

New Aspects of Invasive Growth Regulation Identified by Functional Profiling of MAPK Pathway Targets in Saccharomyces cerevisiae

“…Rho5p is a small GTPase of the Rho family (GARCIA-RANEA AND VALENCIA 1998;SINGH et al 2008;SCHMITZ et al 2018;SINGH et al 2019). RHO5 was not a target of the fMAPK pathway, however, it is regulated by the GTPase-activating protein Rgd2p (ANNAN et al 2008), which was a target of the fMAPK pathway (Fig.…”

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

“…As the downstream MAP kinases in both the CWI and the HOG signalling pathway were shown to be required for mitophagy [ 18 , 19 ], it seems plausible that Rho5 at least partially acts by modulating their activity. A more direct role in mitophagy was also suggested from high-throughput screens, which identified Atg21 and Msp1, components of the mitophagy pathway and the outer mitochondrial membrane, respectively, as interaction partners of Rho5 [ 20 ].…”