Glutaredoxins Grx4 and Grx3 of Saccharomyces cerevisiae play a role in actin dynamics through their Trx domains, which contributes to oxidative stress resistance

Abstract

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.

FIG. 1.

(A and B) The absence of Grx4 increases cell sensibility to hydrogen peroxide and its overexpression induces cell resistance against the oxidizing agent. All of these effects occur independently of Aft1. The following strains were exponentially grown in SD medium plus amino acids before being serial diluted and spotted onto plates either containing, or not containing, 1 mM H₂O₂. The plates were incubated at 30°C for 3 days. (C) Same as in panels A and B, except that the plates contained 1.75 mM hydrogen peroxide. wt, wild type.

FIG. 2.

The grx3 grx4 double mutant exhibits endogenous constitutive oxidative stress caused by ROS accumulation as a consequence of Aft1 misregulation. Cells were exponentially grown in SD plus amino acids. Aliquots (1 ml) were removed from each culture to quantify ROS levels using the dihydroethidine method described in Materials and Methods. Time points represent 30 and 60 min after the addition of 1 mM hydrogen peroxide to each of the growing exponentially cell cultures. Histograms represent the average of three independent experiments. wt, wild type.

FIG. 3.

Grx3 and Grx4 perform an additive function in remodeling actin cables both under normal conditions and upon treatment with hydrogen peroxide. (A) Actin staining of samples from wild-type and grx3, grx4, and grx3 grx4 mutant cultures treated with 1 mM hydrogen peroxide. Samples were collected for actin staining at the times indicated in the figure. (B) Histograms representing the quantification of panel A. (C) Actin staining of the same samples as in panel A with temperatures shifted from 30 to 38°C for the times indicated. (D) Quantification of the experiment represented in panel C. wt, wild type.

FIG. 4.

Neither iron nor ROS accumulation provoked by Aft1 misregulation causes the actin cytoskeleton disorganization observed in the grx3 grx4 double mutant. (A) Actin staining of samples from the wild-type and grx3 grx4 and grx3 grx4 aft1 mutant cultures either treated, or not treated, with 1 mM hydrogen peroxide. (B) Quantification of the experiment shown in panel A. wt, wild type.

FIG. 5.

Elevated concentration of intracellular iron and hydroxyl radicals are not sufficient to explain actin disorganization in the grx3 grx4 mutant. Wild-type and grx3 grx4 mutant strains were logarithmically grown and treated with 2 mM ferrozine for 6 h. Samples were collected at time zero and after 6 h of treatment for actin staining (A) and to quantify the levels of superoxide (B). (C) Exponentially growing wild-type and grx3 grx4 mutant cells were grown in the presence of the ROS scavenger Mn²⁺. Samples were treated with 2 mM Mn₂SO₄ for 2 h. Samples were collected for actin staining (C) and to quantify the levels of superoxide (D). Histograms represent the averages of three independent experiments. wt, wild type.

FIG. 6.

Overexpression of Grx3 compensates for the lack of Grx4 function in the restoration of actin cytoskeleton polarity and in cell viability, both under normal and oxidative conditions. (A) Actin staining of exponentially growing cells of grx3 grx4, grx3 grx4+pGrx3, and grx3 grx4+pGrx4. Cultures were either treated, or not treated, with hydrogen peroxide, and samples were collected for actin staining at the times indicated. (B) Quantification of the experiment depicted in panel A. The histograms represent the number of cells without visible cables. (C) Western blot analysis performed with anti-HA polyclonal antibody to detect the desired protein in total protein extracts. We used anti-α-tubulin as a loading control (see Materials and Methods). Cultures from the wild-type and grx3 grx4 mutant strains, transformed with each of the plasmids containing either tetO₇GRX3 or tetO₇GRX4 were grown in SD media to exponential phase (0) and subsequently treated with 1 mM H₂ O₂ for 3 h (3 h). (D) Actin staining of grx4 and grx4+pGrx3 cultures, as in panel A. (E) Quantification of the experiment depicted in panel C, as in panel B. (F) Serial dilutions from exponentially growing cultures of the different strains stated in the figure. SD plates plus the required amino acids either containing or not containing 1 mM hydrogen peroxide were used. Plates were grown at 30°C for 3 days.

FIG. 7.

The Trx domains of Grx3 and Grx4 are sufficient to induce actin cable formation either in grx4 or in grx3 grx4 mutants. (A) Actin staining of the grx4 mutant transformed, or not transformed, with each of the Grx or Trx domains of proteins Grx3 and Grx4, both in logarithmic growth and in response to hydrogen peroxide treatment at the times indicated. (B) Histograms representing statistical values for the experiment performed in panel A. (C) Same as in panel A, but with the grx3 grx4 double mutant. (D) Quantification of the experiment performed in panel C.

FIG. 8.

The Trx domains of Grx3 and Grx4 are involved in cell survival under conditions of oxidative stress. grx4 (A) and grx3 grx4 (B) mutants were transformed with the Grx and Trx domains of Grx3 and Grx4. Exponentially growing cells of the different cultures from this figure were serial diluted and plated onto plates either containing, or not containing, 1 mM hydrogen peroxide. Plates were grown at 30°C for 3 days.

FIG. 9.

Each of the Grx domains of either Grx3 or Grx4 favor ROS detoxification in grx3 grx4 mutant cells. The grx3 grx4 mutant and wild-type strains were either transformed, or not transformed, with each of the plasmids, which alternatively contained the Trx or Grx domains of either Grx3 or Grx4 proteins. Exponentially growing cultures were either treated, or not treated, with hydrogen peroxide to determine the anion superoxide concentration in the cells, which was represented in units of fluorescence. Histograms represent averages of three independent experiments. wt, wild type.