Grx3 and Grx4, two monothiol glutaredoxins of Saccharomyces cerevisiae, regulate Aft1 nuclear localisation. We provide evidence of a negative regulation of Aft1 activity by Grx3 and Grx4. The Grx domain of both proteins played an important role in Aft1 translocation to the cytoplasm. This function was not, however, dependent on the availability of iron. Here we demonstrate that Grx3, Grx4 and Aft1 interact each other both in vivo and in vitro, which suggests the existence of a functional protein complex. Interestingly, each interaction occurred independently on the third member of the complex. The absence of both Grx3 and Grx4 induced a clear enrichment of G1 cells in asynchronous cultures, a slow growth phenotype, the accumulation of intracellular iron and a constitutive activation of the genes regulated by Aft1. The grx3grx4 double mutant was highly sensitive to the oxidising agents hydrogen peroxide and t-butylhydroperoxide but not to diamide. The phenotypes of the double mutant grx3grx4 characterised in this study were mainly mediated by the Aft1 function, suggesting that grx3grx4 could be a suitable cellular model for studying endogenous oxidative stress induced by deregulation of the iron homeostasis. However, our results also suggest that Grx3 and Grx4 might play additional roles in the oxidative stress response through proteins other than Aft1.

In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15_ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast. Cytokinesis requires assembly of an actomyosin ring adjacent to the plasma membrane, which upon contraction pulls the membrane to form a cleavage furrow. In fungi ring closure is coordinated with the synthesis of a cell wall septum. Knowledge about the molecules anchoring the ring to the membrane is very limited. We have found that fission yeast paxillin, located at the ring, and Bgs1, the enzyme responsible for primary septum formation, located at the membrane, cooperate during cytokinesis. Both are required to anchor the ring to the membrane and to maintain it during cytokinesis. Moreover, both proteins cooperate to form the septum. Accordingly, paxillin is essential when Bgs1 is depleted. When both proteins are missing, the contractile ring forms but the lateral cell wall overgrows inwards without a defined cleavage furrow and septum formation. During cytokinesis there is an increase of paxillin which depends on the SH3 domain of the F-BAR protein Cdc15. Consequently the absence of this domain mimics the phenotype of paxillin absence in Bgs1-depleted cells. Interestingly, a decreased function of both Cdc15 and paxillin uncouples the septum synthesis from the ring contraction, indicating an essential cooperation between these proteins and Bgs1 for proper cytokinesis.

Mtl1 is a member of the cell wall integrity (CWI) pathway of Saccharomyces cerevisiae, which functions as a cell wall sensor for oxidative stress. Genome-wide transcriptional analysis revealed a cluster of genes that were down-regulated in the absence of Mtl1. Many of these genes were potentially regulated by the general stress response factor Msn2/Msn4. In response to rapamycin, caffeine, glucose starvation and oxidative stress provoked by H₂O₂, mtl1 presents a significant loss of viability as well as a deficiency in the transcriptional response mediated by Msn2/Msn4. The Mtl1 function was required (i) to induce ribosomal gene repression, (ii) to induce the general stress response driven by the transcription factor Msn2/Msn4, and (iii) to activate the CWI pathway in response to both glucose starvation and oxidative stress. We also detected higher cAMP levels in the mtl1 mutant than in wild type cells indicative of up-regulated RAS2-PKA activity. Disruption of TOR1, disruption of RAS2, or hyperactivation of Rho1 restored both the viability and the transcriptional function (both ribosomal and Msn2/Msn4-dependent gene expression) in the mtl1 mutant to almost wild type levels when cells were starved of glucose or stressed with H₂O₂. Taking our results together, we propose an essential role for Mtl1 in signaling oxidative stress and quiescence to the CWI pathway and to the general stress response through Rho1 and the inhibition of either the TOR1 or RAS2 functions. These mechanisms would be required to allow cells to adapt to both oxidative and nutritional stresses.

Grx3 and Grx4 are two monothiol glutaredoxins of Saccharomyces cerevisiae that have previously been characterized as regulators of Aft1 localization and therefore of iron homeostasis. In this study, we present data showing that both Grx3 and Grx4 have new roles in actin cytoskeleton remodeling and in cellular defenses against oxidative stress caused by reactive oxygen species (ROS) accumulation. The Grx4 protein plays a unique role in the maintenance of actin cable integrity, which is independent of its role in the transcriptional regulation of Aft1. Grx3 plays an additive and redundant role, in combination with Grx4, in the organization of the actin cytoskeleton, both under normal conditions and in response to external oxidative stress. Each Grx3 and Grx4 protein contains a thioredoxin domain sequence (Trx), followed by a glutaredoxin domain (Grx). We performed functional analyses of each of the two domains and characterized different functions for them. Each of the two Grx domains plays a role in ROS detoxification and cell viability. However, the Trx domain of each Grx4 and Grx3 protein acts independently of its respective Grx domain in a novel function that involves the polarization of the actin cytoskeleton, which also determines cell resistance against oxidative conditions. Finally, we present experimental evidence demonstrating that Grx4 behaves as an antioxidant protein increasing cell survival under conditions of oxidative stress.

Oxidative stress causes transient actin cytoskeleton depolarization and also provokes vacuole fragmentation in wild-type cells. Under conditions of oxidative stress induced by hydrogen peroxide, the Slt2 protein is required to repolarize the actin cytoskeleton and to promote vacuole fusion. In this study, we show that grx3 grx4 and grx5 mutants are cellular models of endogenous oxidative stress. This stress is the result of alterations in iron homeostasis that lead to impairment of vacuolar function and also to disorganization of the actin cytoskeleton. Slt2 overexpression suppresses defects in vacuolar function and actin cytoskeleton organization in the grx3 grx4 mutant. Slt2 exerts this effect independently of the intracellular levels of reactive oxygen species (ROS) and of iron homeostasis. The deletion of SLT2 in the grx3 grx4 mutant results in synthetic lethality related to vacuolar function with substantial vacuole fragmentation. The observation that both Vps4 and Vps73 (two proteins related to vacuole sorting) suppress vacuole fragmentation and actin depolarization in the grx3 grx4 slt2 triple mutant strengthens the hypothesis that Slt2 plays a role in vacuole homeostasis related to actin dynamics. Here we show that in sod1 , grx5 , and grx3 grx4 slt2 mutants, all of which are affected by chronic oxidative stress, the overexpression of Slt2 favors vacuole fusion through a mechanism dependent on an active actin cytoskeleton.