Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Abstract

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.

FIG. 1

Comparative analysis of glutaredoxin sequences. (A) Alignment of the S. cerevisiae Grx3, Grx4, and Grx5 amino acid sequences deduced from the nucleotide sequences of their respective ORFs. Common residues in the three sequences are shaded. The N-terminal extensions of Grx3 and Grx4 are not represented. The asterisk marks the common cysteine residue present in all three sequences. A second cysteine present in Grx5 is underlined. (B) Sequence analysis of relevant regions of 23 different glutaredoxin proteins. Regions N and C are respectively the most N- and C-terminal regions of the molecules for which significant alignments can be established. Sequences outside these two regions are not represented. Subfamilies 1 and 2 are initially defined according to the consensus sequences indicated in the figure. For the consensus sequences, residues identical in all members of each subfamily are represented in uppercase letters, while those common to at least 75% of them are in lowercase letters. More details about these sequences can be obtained from reference . H. ducreyi, Haemophilus ducreyi; H. influenzae, Haemophilus influenzae; L. pneumophila, Legionella pneumophila; R. prowazekii, Rickettsia prowazekii; C. elegans, Caenorhabditis elegans.

FIG. 2

Sequence Space analysis of the glutaredoxin family. Principal component analyses of the protein sequences are shown (from left to right) on the resulting 1-2, 1-3, and 2-3 discriminant axes (8). Analyses were carried out separately for region N in subfamily 1, region N in subfamily 2, and region C in the whole glutaredoxin family. Each point in the plots represents an individual sequence identified by a number. Distances between points are proportional to sequence divergence. Sequence clusters are defined according to proximity in the resulting plots (continuous lines). These clusters were tentatively divided into subsets of sequences (dashed lines) when the results on the three dimensions suggested the existence of relevant subgroups. See the Fig. 1 legend for genus abbreviations.

FIG. 3

Protein oxidative damage under normal growth conditions of wild type and grx single and double mutants. MATa strains were employed. Cultures of wild type (CML235) and single and double mutants were grown in YPD liquid medium at 30°C until an optical density at 600 nm of 1 was reached. The crude extracts obtained were analyzed by Western blotting with anti-DNP antibodies (B). A parallel run stained with Coomassie brilliant blue is shown in panel A. Each lane contained 20 μg of total protein. Asterisks mark the identified transketolase band (see text for details).

FIG. 4

Sensitivity of S. cerevisiae grx mutants to oxidative agents. MATa strains were employed. (A) Cultures of wild-type (CML235) and single mutant strains growing exponentially in YPD liquid medium at 30°C were exposed to the indicated agents and concentrations, and viable numbers (relative to time zero values) were determined at different times. (B) As in panel A, except that lower agent concentrations were used to determine sensitivity of double mutants compared to wild-type and single mutant strains. (C) Protein oxidative damage in wild type and glutaredoxin mutants under stress conditions. Cultures of wild type and single glutaredoxin mutants were grown in YPD liquid medium at 30°C, and at an optical density at 600 nm of 1, menadione or hydrogen peroxide was added to the cultures at the final concentration of 20 or 5 mM, respectively. After 60 min of treatment, the cultures were harvested by centrifugation and crude extracts were obtained. Analyses by Western blotting with anti-DNP antibodies were conducted as described in Materials and Methods. Each lane contained 10 μg of total protein.

FIG. 5

Sensitivity of S. cerevisiae grx mutants to hyperosmotic treatments. (A) Exponentially growing wild-type (CML235) and mutant (MATa type) cells in YPD medium at 30°C were supplemented with 2 M KCl, and cell viability (made relative to parallel untreated cultures) was determined at the indicated times. (B) Exponentially growing cells in YPD medium were treated with sorbitol at the final concentrations indicated, and incubation was continued under these conditions. Total cell numbers were measured at subsequent periods. Bars represent the lag periods after sorbitol addition during which cell division remained arrested before cultures resumed growth.

FIG. 6

Effect of oxidative stress (5 mM hydrogen peroxide for 1 h) on cell viability of grx mutants (MATa strains) compared to that of wild-type cells (strain CML235). Cells were grown exponentially at 30°C in SD medium plus glucose, and after treatments, they were plated on YPD solid medium in order to determine viability. Bars indicate the percentages of viable cells relative to those in parallel untreated cultures.

FIG. 7

Northern blot analyses of GRX3, GRX4, and GRX5 expression. Samples were taken at different stages of the population growth curve in YPD liquid medium at 30°C (A) or after treatment of mid-exponential-phase cells (at 30°C except for heat shock) with KCl (0.5 M), hydrogen peroxide (0.4 mM), menadione (2 mM), or heat shock for the indicated times (B). Small nuclear U2 mRNA is shown as the loading control. Numbers under the lanes indicate the mRNA levels for each time point, relative to the mid-exponential-phase sample. For heat shock analysis, the time zero sample corresponds to exponential cultures at 25°C.