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Vol. 15, Issue 10, 4522-4531, October 2004

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Old Yellow Enzyme Protects the Actin Cytoskeleton from Oxidative Stress

Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, NY 13210

Submitted June 3, 2004; Accepted July 27, 2004
Monitoring Editor: David Drubin

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Old Yellow Enzyme (OYE) has long served as a paradigm for the study of flavin-containing NADPH oxido-reductases and yet its physiological role has remained a mystery. A two-hybrid interaction between Oye2p and actin led us to investigate a possible function in the actin cytoskeleton. We found that oye deletion strains have an overly elaborate actin cytoskeleton that cannot be attributed to changes in actin concentration but likely reflect stabilization of actin filaments, resulting in excessive actin assembly. Cells expressing the actin mutant act1-123p, which has a weakened interaction with Oye2p, show comparable defects in actin organization to the oye deletion strain that can be suppressed by overexpression of Oye2p. Similarly, mutation of either conserved cysteine of the potential disulfide pair Cys285-Cys374 in actin completely suppresses the actin organization defect of the oye phenotype. Strains lacking Oye function are also sensitive to oxidative stress as induced by H₂O₂, menadione, and diamide treatment. Mutation of either Cys285 or Cys374 of actin suppresses the sensitivity of oye strains to oxidative stress and in fact confers super-resistance to oxidative stress in otherwise wild-type strains. These results suggest that oxidative damage to actin, like that which has been observed in irreversibly sickled red blood cells, may be a general phenomenon and that OYE functions to control the redox state of actin thereby maintaining the proper plasticity of the actin cytoskeleton. In addition to uncovering a long sought biological function for Old Yellow Enzyme, these results establish that cellular sensitivity to oxidative stress can in part be directly attributed to a specific form (C285-C374 disulfide bond formation) of oxidative damage to actin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Old Yellow Enzyme (OYE) has a long investigative history beginning with its identification by the German biochemists Warburg and Christian from brewer's bottom yeast in 1933 (Warburg and Christian, 1933). Since that time, OYE has served as a model for enzymologists interested in enzymecatalyzed redox reactions. Collectively, these investigations have led to a sophisticated understanding of OYE's ability to accept electrons from NADPH onto a flavin mononucleotide cofactor and transfer these electrons to diverse ligands (Schopfer and Massey, 1991). Unfortunately, identification of the physiologically relevant substrate(s) of OYE has been difficult and elusive. Our serendipitous identification of Oye2p as an actin-interacting protein in a two-hybrid screen suggested that OYE might control the redox state of actin.

Many studies on oxidative stress have shown that Cys374 of actin is highly reactive to oxidizing agents and that oxidative damage elicits changes in actin cytoskeketon organization. It has been proposed that oxidative damage can induce intermolecular disulfides that can cross-link actin filaments (for review, see Dalle-Donne et al., 2001). Oxidation of actin has been directly observed in the erythrocyte cytoskeleton of patients with sickle cell anemia (Bencsath et al., 1996). The red blood cell membrane cytoskeleton consists of short (14-subunit-long) actin filaments attached at their ends to spectrin tetramers that are in turn anchored to the plasma membrane via membrane spanning glycoproteins. The actin filaments of this array must be dynamic for the red blood cell to undergo the cell shape changes required to pass through small capillaries. Crisis in sickle cell patients leads to severe pain, organ damage, and mortality and is correlated with the accumulation in the bloodstream of irreversibly sickled red blood cells (ISCs) (Kaul et al., 1983). ISCs have been found to contain hyperstable membrane cytoskeletons compared with normal red blood cells (RBCs) or reversibly sickled red blood cells (RSCs) (Lux et al., 1976). This lack of plasticity in the membrane cytoskeleton contributes to the blockage of small capillaries in turn leading to poor peripheral circulation and organ damage (Fabry et al., 1992). Stabilization of the ISC membrane cytoskeleton is due to the accumulation of oxidized actin (Shartava et al., 1995) that contains an intramolecular Cys285-Cys374 disulfide bond (Bencsath et al., 1996). Filaments formed containing this form of actin show reduced rates of disassembly in vitro (Shartava et al., 1997). Previously, it had been thought that the formation of a C285-C374 disulfide bond in actin was a unique result of the extremely oxidizing environment in RBCs exacerbated in sickle cell RBCs by spilling of iron from the mutant hemoglobin and decreases in cellular reductants such as reduced glutathione (Wetterstroem et al., 1984).

Saccharomyces cerevisiae has been a particularly good model system for investigating how cells protect themselves from oxidative stress resulting from both metabolic activity and environmental insult. These studies have led to the view that the glutaredoxin and thioredoxin systems are largely responsible for repairing oxidative damage to cytosolic proteins (for review, see Wheeler and Grant, 2004). The results reported here suggest that S. cerevisiae actin is susceptible to the same oxidative damage as red blood cell actin and that like ISC red blood cells, disulfide bond formation in actin is a major source of the physiological response and sensitivity to oxidative stress. The roles of the glutaredoxin or thioredoxin systems in repairing oxidative damage to actin are currently unclear, but our results strongly argue that Old Yellow Enzyme is recruited to repair the Cys285-Cys374 disulfide bond of oxidized actin.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cell Culture, Construction of Strains and Plasmids
Yeast strains are listed in Table 1. The actin alleles used in this study are described in Table 2. Standard methods were used for growth, sporulation, and genetic analysis of yeast (Rose et al., 1989).

Double fusion polymerase chain reaction (PCR) (Amberg et al., 1995b) was used to disrupt both the OYE2 and OYE3 genes. Codons 37–388 of OYE2 were removed and replaced with the HIS3 gene, whereas codons 75–391 of OYE3 were removed and replaced with the TRP1 gene.

The codons for C285 and C374 of actin were converted to alanine codons by overlap/fusion PCR (Ho et al., 1989). An ACT1 allele marked with a HIS3 gene (Wertman et al., 1992) was used as a template for the PCR reactions that were then integrated into the genome of wild-type diploid strain FY23x86. The mutations were confirmed by sequencing, all mutants were constructed in duplicate from two separate PCR reactions, and the results reported are for derivatives of both isolates.

Primer Dao-OYE2-30 (5'-CTTCCGTAATGAACTTCCGTA-3') and primer Dao-OYE2–31 (5'-GCCGGAATTCTAAATCTGTTGCTGCCCAAGC-3') were used to PCR amplify an 1819-base pair fragment of the OYE2 genomic locus from S288c background, genomic DNA. The fragment was digested with EcoRI and BglII and cloned into EcoRI/BamHI digested YCplac33. The entire OYE2 insert was confirmed by DNA sequencing and was then subsequently subcloned in two steps into the PstI and XbaI sites of the 2 µ/LEU2 vector YEp351.

Two-Hybrid Analysis
Two-hybrid screening was performed using a GAL4 DNA binding domain (DBD)-actin fusion against a yeast cDNA library as described previously (Amberg et al., 1995a). Two-hybrid analysis of actin and oye2 alleles was performed by transforming yeast strain Y190 with contrasts expressing the GAL4 DBD fusions and yeast strain Y187 with contrasts expressing the GAL4 activation domain (AD) fusions, mating the transformants, selecting diploids that carry both plasmids on synthetic complete medium lacking leucine and tryptophan, and assaying for activation of the HIS3 reporter on synthetic medium containing 10 µg/ml adenine and 25 mM 3-aminotriazole (Sigma-Aldrich, St. Louis MO).

Actin Expression Analysis (Table 2)
Cells were grown to 4 x 10⁷/ml, washed in phosphate-buffered saline (PBS), and lysed in a French Press (American Instrument, Silver Spring, MD) at 1200 pounds per square inch. The extract was clarified at 12,000 rpm for 20 min in a Beckman JA20 rotor. Protein concentrations in the extracts were determined by a Bradford assay. Protein (1.7 µg) from each extract was separated by 10% SDS-PAGE, transferred to nitrocellulose, blotted with C4 mouse anti-actin antibody (Sigma-Aldrich) at a 1:500 dilution and with goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Sigma-Aldrich) at a 1:2000 dilution and visualized by chemiluminescence (Pierce Chemical, Rockford, IL).

Microscopy
Cells were fixed with 4% formaldehyde, stained with rhodamine-phalloidin (Molecular Probes, Eugene, OR) as described previously (Pringle et al., 1989), and visualized by differential interference microscopy and/or fluorescence microscopy on a Zeiss Axioskop 2 MOT (Carl Zeiss, Jena, Germany) by using a Plan-APOCHROMAT 100x/1.4 numerical aperture objective. Images were captured with an Orca ER camera (Hamamatsu, Malvern, PA) into OpenLab software (Improvision, Lexington, MA) and manipulated in Adobe Photoshop (Adobe Systems, San Jose, CA).

Flow Cytometry
Cells were fixed with 4% formaldehyde (by addition to the growth medium) for 10 min. The cells were then collected and resuspended in PBS + 4% formaldehyde and incubated for 1 h. The cells were washed twice with PBS, and 4 x 10⁷ cells were stained with 100 µl of fluorescein isothiocyanate (FITC)-phalloidin (Molecular Probes) in a 1-ml total volume for 1 h. The cells were washed four times with PBS and suspended in 1 ml of PBS. Fluorescence-activated cell sorting (FACS) analysis was performed on a FACStar Plus (BD Biosciences, San Jose CA) set to a 488-nm excitation and 530-nm band pass. Means calculated are for the average intensity in the peak of FITC-phalloidin–stained cells and the coefficient variable for the peak, or width of the peak at half height, also was calculated.

Latrunculin A (LatA) Halo Assays
Ten microliters of stationary phase culture was mixed into 2 ml of 2x YPD plus 2 ml of 1% agar at 55°C. The agar suspension was poured onto the surface of a YPD plate and allowed to solidify. Sterile Whatman paper filter discs were placed on the agar surface, and 10 µl of latrunculin A (gift of P. Crews, University of California, Santa Cruz, Santa Cruz, CA) diluted to 1, 1.5, and 2 mM in dimethyl sulfoxide was spotted on the discs. The plates were incubated at 30°C for 20 h before being photographed.

Sensitivity to Oxidative Stress
Ten microliters of stationary phase culture was mixed into 2 ml of 2x YPD plus 2 ml of 1% agar at 55°C. The agar suspension was poured onto the surface of a YPD plate and allowed to solidify. Sterile Whatman paper filter discs were placed on the agar surface, and 10 µl of H₂O₂ diluted to 1, 2, 5, and 10% in H₂O or 10 µl of menadione sodium bisulfite (Sigma-Aldrich) diluted to 5, 10, 20, and 40 mM in H₂O or 10 µl of diamide (N,N,N',N'-tetramethylazodicarboxamide; Sigma-Aldrich) diluted to 50, 100, 250, and 500 mM in H₂O was spotted on the discs. The plates were incubated at 30°C for 3–4 d before being photographed.

For growth curves and actin staining in H₂O₂-treated cells, wild-type strain FY86 and oye2 oye3 strain DAY127 were grown to 1 x 10⁷ cells/ml at which time H₂O₂ was added to 0.01%. Growth was monitored by taking optical density measurements in a Klett Meter (Bel-Art Products, Pequannock NJ) over time. Fixation for rhodamine-phalloidin staining was initiated 480 min after H₂O₂ addition but was otherwise as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Analysis of the Actin–Oye2p Interaction
A clone encoding the C terminus of Oye2p (amino acids 259–400, plasmid pAIP7) fused to the GAL4 AD of plasmid pACT (Durfee et al., 1993) was found in a two-hybrid screen to cause robust activation of two-hybrid reporters when coexpressed in yeast with a fusion of yeast actin to the GAL4 DNA binding domain (plasmid pDAb7; Figure 1A). Note that the two-hybrid strains carry an ade2 mutation that causes the accumulation of a red pigment in the growing cells. This assists in the visualization of those cells that are growing, thereby indicating a positive two-hybrid interaction on the selective medium. The importance of the C terminus of Oye2p (displayed in red on Oye2p in Figure 1B) for the interaction was confirmed by deleting the last three codons of OYE2 (for Asp Lys Asn; displayed in blue on Oye2p in Figure 1B) from plasmid pAIP7 (creating plasmid pJH2) and showing that the interaction with actin is lost (see right-most column in Figure 1A). The actin–Oye2p interaction was further investigated with 35 alanine scan mutants of actin (Wertman et al., 1992) of which three (act1–119, act1–123, and act1–134) were found to be specifically defective for interacting with Oye2p (the corresponding amino acids are displayed in red on human -actin in Figure 1C; note that the residues changed by act1-134 are buried). The codon changes in these alleles are described in Table 2.

View larger version (41K): [in this window] [in a new window]   Figure 1. The C terminus of Oye2p interacts with actin in the vicinity of a potential C285-C374 disulfide bond. (A) Strains expressing fusions to the GAL4 DBD were mated to strains expressing fusions of Oye2p to the GAL4 AD. Activation of the HIS3 two-hybrid reporter was assessed by growth on plate medium lacking histidine and containing the His3p inhibitor 3-aminotriazole. (B) The solvent-accessible surface of reduced Oye2p (Fox and Karplus, 1994) was calculated and displayed with the program VMD (Humphrey et al., 1996). The C-terminal region first identified as interacting with actin in a two-hybrid screen is colored in red (amino acids 259–400). The three C-terminal residues (D398, K399, and N400) shown to be required for the actin interaction are colored in blue. The FMN cofactor is labeled and colored in yellow. (C) The solvent-accessible surface of human -actin from the actin-profilin cocrystal structure (Schutt et al., 1993) is shown. Mutations that disrupt the actin–Oye2p two-hybrid interaction are labeled by allele numbers and colored red. Cysteines 285 and 374, known to form a disulfide in ISC actin, are labeled and colored yellow.

The mutational analysis suggested that Oye2p interacts with actin in proximity to the C285-C374 disulfide bond (these cysteines are displayed in yellow on actin in Figure 1C) observed in red blood cell ISC actin (Bencsath et al., 1996). The data are modeled on actin derived from an actinprofilin cocrystal structure (Schutt et al., 1993) because this is the only structure of unmodified actin in which the C-terminal cysteine has been assigned. Unfortunately, in the profilactin crystal structure the cysteines seem distant from each other. Nevertheless, these data indicate that Oye2p could be positioned such that its flavin mononucleotide (FMN) cofactor (displayed in yellow in Figure 1B) would be in proximity to the disulfide (displayed in yellow on actin in Figure 1C).

S. cerevisiae has a homologue of OYE2 called OYE3 (Niino et al., 1995). However, a comparable construct expressing Oye3p was unable to interact with actin. In addition, Oye3p did not seem to be expressed under standard growth conditions in our strain background. However, an examination of recently published genome sequences of related yeasts (Kellis et al., 2003) shows that OYE3 is conserved, indicating it is a bona fide gene.

Visualization of Actin Organization Defects in oyedeficient Cells
OYE2 had previously been shown to be nonessential (Stott et al., 1993). Constructing a nearly complete knock out (codons 37–388 were deleted in our strain background) reconfirmed this, although a slight growth deficit was observed. A deletion allele of OYE3 also was constructed, and a genetic cross was performed to construct the viable oye2 oye3 strains DAY127 and DAY128. This was done to avoid any potential contribution to function from the OYE3 locus. Western assays with high-affinity antibodies failed to detect immunoreactive peptides in this strain, confirming the absence of Oye proteins. To aid in microscopic analysis (diploids are larger than haploids), double deletion strains were mated to form the diploid strain DAY127x128, and its actin cytoskeleton was visualized by rhodamine-phalloidin staining and fluorescence microscopy (Figure 2). Compared with the congenic wild-type control FY23x86 (Figure 2A), the oye cells displayed an overly elaborate actin cytoskeleton with excessive quantities of actin cables and actin cortical patches (Figure 2B). Accompanying the actin defects were striking aberrations in cell morphology, including excessively large cells, elongated cells and hyperelongated buds (Figure 2B, arrow). The actin staining suggests that the cell morphology defects are likely caused by a failure of the cells to accurately control polarized cell growth. Note that in comparable analyses, oye3 strains were undistinguishable from wild-type controls (our unpublished observations).

View larger version (50K): [in this window] [in a new window]   Figure 2. Loss of Oye or weakening Oye2p's interaction with actin leads to excessive actin assembly in vivo. Logarithmically growing diploid wild-type (A), oye2 oye3 (B), and act1-123 (C) cells were stained with rhodamine-conjugated phalloidin and visualized by fluorescence microscopy. Bars, 5 µm.

Although actin overexpression does not induce a similar phenotype to the oye strain (our unpublished observations), to preclude the possibility that increases in actin expression are contributing to actin cytoskeleton elaboration in the oye strain we examined actin expression levels. Extracts were prepared from wild-type and oye strains, and serial dilutions of equivalent amounts of protein were analyzed in a Western blot with anti-actin antibodies. At all dilutions (only one dilution is shown in Figure 3), the wildtype strain (lane 1) expressed a very similar amount of actin to the oye strain (lane 2), indicating that the apparent increase in F-actin structures in the oye strain probably results from a skewing of the F-to G-actin ratios.

View larger version (34K): [in this window] [in a new window]   Figure 3. Actin levels are unaffected in strains defective for Oye function. Total cellular protein (1.7 µg) from wild-type strain FY86 (lane 1), oye2oye3 strain DAY127 (lane 2), and act1-123 strain TDS154 (lane 3) was analyzed in a Western assay with anti-actin antibodies.

The Oye2p–Actin Interaction Is Required for Normal Actin Cytoskeleton Organization
The diploid strain TDS154x454, homozygous for the act1-123 allele (act1-123p is defective for the Oye2p interaction; Figure 1A), was stained with rhodamine-phalloidin and examined for actin organization defects (Figure 2C). Like the oye strain DAY127x128, the act1-123 cells showed excessive elaboration of the actin cytoskeleton and the accompanying cell morphology defects despite the presence of Oye2p. Although similar in many regards, the act1-123 cultures had a greater percentage of cells with extremely elongated buds than the oye cultures and consistently the F-actin structures in the act1-123 cells stained weakly with rhodamine-phalloidin. Therefore, the act1-123 mutant seems to be a partial phenocopy of the oye mutant, suggesting that the act1-123 protein has defects beyond its defects in interacting with Oye2p. However, the act1-123 and oye strains do share an aspect of their phenotypes (excessive actin cytoskeleton elaboration) that has not been previously observed in yeast loss of function mutants. This suggests that the actin defects of the act1-123 cells are in part due to defects in the inability of Oye2p to interact with the mutant actin and conversely that Oye2p's affects on the actin cytoskeleton are not mediated indirectly but by Oye2p interacting with actin to alter its assembly dynamics. Note that because act1-134 is a lethal allele and act1-119 causes a very pleitropic phenotype, including severe temperature sensitivity, we were unable to use these strains to address the role of the Oye-actin interaction in vivo.

If excessive actin cytoskeleton elaboration in the act1-123 strain is in fact the result of reduced affinity for Oye2p, then overexpression of Oye2p might be expected to suppress this aspect of the act1-123 phenotype. This hypothesis was tested by constructing a 2 µ-based, plasmid encoded allele of the wild-type OYE2 gene (plasmid pBH405). Introduction of this plasmid into a wild-type strain did not induce a discernible mutant phenotype (our unpublished observations). However, this high copy OYE2 plasmid seemed to completely suppress the actin organization and cell morphology defects of the homozygous act1-123/act1-123 strain TDS154x454 (Figure 4B) compared with the same strain transformed with the parent vector backbone YEp351 (Figure 4A).

View larger version (50K): [in this window] [in a new window]   Figure 4. Overexpression of OYE2 suppresses the actin cytoskeleton and cell morphology defects caused by the act1-123 allele. The act1-123 homozygous diploid strain TDS154x454 was transformed with the 2 µ-based vector YEp351 (A) or the same vector carrying the OYE2 gene (plasmid pBH405; B). The cells were stained with rhodamine-phalloidin and visualized by fluorescence microscopy. Bars, 5 µm.

Quantification of Actin Defects in oye-deficient Cells
Actin organization defects in oye and act1-123 cells were quantified in two manners. First, actin structures in 10 rhodamine-phalloidin–stained diploid cells of each strain were visually counted. The cells were chosen to be at approximately the same stage of the cell cycle by analyzing cells with buds approximately one-third the size of the mother cell body. The numbers reported are averages with standard deviations for 10 cells (Table 3). Contiguous actin cables were counted as well as patches in the bud and mother cell body. The oye2 oye3 cells contained significantly higher numbers of actin cables and patches in the bud. The oye2 oye3 cells also had a higher average number of cortical patches in the mother than wild-type but this attribute seemed to be highly variable in the oye strain and was therefore not strictly statistically significant. The act1-123 cells displayed a comparable elevation to the oye strain in actin cables and less so for cortical patches. Note that the very light rhodamine-phalloidin staining in the act1-123 cells made counting actin structures difficult and so it is possible that the numbers reported are an underestimate.

Second, FITC-phalloidin–stained haploids were analyzed by flow cytometry to measure fluorescence per cell. For 30,000 cells analyzed, two populations were identified: a small percentage of unstained cells and cells that displayed a fluorescein signal well above that of unstained control cells. The mean signal was calculated for the stained cells as a measure of fluorescein staining, and the width of the peak at one-half height was calculated as a measure of the variability of the stained cells (Table 3). The oye3 strain was very similar to the wild-type control in agreement with microscopic inspection of rhodamine-phalloidin–stained cells (our unpublished observations). However, in multiple experiments (the results of one representative experiment are shown in Table 3) the oye2 cells displayed a higher fluorescent signal (30% higher) than wild-type controls. The oye2 oye3 strain was comparable to the oye2 strain but did show greater variability than the wild-type strain in agreement with the morphological heterogeneity of the oye2 oye3 strains as observed microscopically. We conclude that oye2-deficient, but not oye3-deficient cells, contain higher amounts of F-actin structures than the wild-type control. As previously observed under the microscope, the act1-123 cells stained poorly in multiple experiments (see comment in section below) and yet, as described above, contain an overabundance of F-actin structures. Therefore, FACS analysis of the act1-123 strain is not useful for quantifying F-actin structures.

The Actin Cytoskeleton of oye-deficient Cells Is More Stable
Excessive F-actin elaboration in the oye cells in the absence of heightened actin expression suggests that the oye cells are either hyperactive for F-actin assembly or assemble a more stable actin cytoskeleton. To test the latter possibility, we asked whether oye cells have altered sensitivities to the G-actin binding and sequestering drug LatA. Halo assays were performed in which different concentrations of LatA were spotted on filter paper discs sitting on top agar containing suspensions of yeast cells (Figure 5). In several experiments (scored blindly by laboratory mates), the oye2 oye3 strain (Figure 5B) displayed resistance to LatA over that observed in the wild-type control (Figure 5A), suggesting that the oye strain does in fact have a more stable actin cytoskeleton. As can be seen the effect is subtle but one might expect that even small changes in filament stability could lead to the dramatic alterations in actin cytoskeleton appearance reported in Figure 2 above. In contrast, we found that the act1-123 strain is extremely sensitive to latrunculin A as reported previously (Ayscough et al., 1997). Mutations in the nucleotide binding cleft have been found to affect both LatA and phalloidin binding (Ayscough et al., 1997; Belmont et al., 1999). Because the act1-123 allele also changes residues in the cleft, it seems likely that both the LatA sensitivity and poor phalloidin staining of act1-123 cells reflect altered affinities of these drugs for act1-123p. Furthermore, because the LatA sensitivity of the act1-123 strain could not be suppressed by overexpression of Oye2p (our unpublished observations), we believe that the LatA sensitivity of the act1-123 mutant is unrelated to defects in the actin–Oye2p interaction.

View larger version (91K): [in this window] [in a new window]   Figure 5. OYE-deficient cells are resistant to latrunculin A. Halo assays were performed to determine the sensitivity of wild-type strain FY23x86 (A), oye2 oye3 strain DAY127x128 (B), and act1-123 strain TDS154x454 (C) to different concentrations of latrunculin A.

Cytoskeletal Defects in oye-defective Strains Can Be Suppressed by Blocking C285-C374 Disulfide Bond Formation in Actin
The enzymatic characteristics of OYE and the proximity of its site of interaction to cysteines 285 and 374 (as shown in Figure 1C) suggested that OYE might have a role in regulating a disulfide between these residues. This was examined by singly mutating Cys285 (act1-201) and Cys374 (act1-200) to alanine. Strains carrying these alleles were viable; grew well at 25, 30, or 37°C, and had a normal cell morphology. The act1-201 strain had a completely normal actin cytoskeleton as determined by rhodamine-phalloidin staining but was sensitive to LatA. In contrast, the act1-200 strain displayed wild-type sensitivity to LatA and an interesting but subtle defect in actin organization: in many cells the actin cables only extend a short distance from the neck into the mother cell body (for an example, see arrows on Figure 6B). These alleles were crossed into an oye background, the resulting progeny were diploidized (strains DAY175 x 176 and DAY173 x 174) and examined by rhodamine-phalloidin staining and differential interference microscopy (Figure 6). Both the act1-200 and act1-201 alleles fully suppressed the excessive actin organization (Figure 6, B and C), and cell morphology defects (Figure 6, E and F) conferred by the absence of OYE (Figure 6, A and D). Note that the actin defects associated with the act1-200 (short cables in the mother; Figure 6B) and act1-201 (LatA sensitivity; our unpublished data) also were observed in the double mutant strains. Therefore, it is formally possible that each allele is suppressing the loss of Oye by a different mechanism but it seems much more likely that they both suppress because neither mutant is capable of forming the C285 to C374 disulfide bond.

View larger version (92K): [in this window] [in a new window]   Figure 6. The C285A and C374A mutations suppress the actin defects of an oye strain. Cells from logarithmically growing oye2 oye3 diploid strain DAY127x128 (A and D), oye2 oye3 act1-200 (C374A) diploid strain DAY175x176 (B and E), and oye2 oye3 act1-201 (C285A) diploid strain DAY173x174 (C and F) were stained with rhodamine-phalloidin (A–C) and visualized by fluorescence microscopy. (D–F) Parallel cultures were examined by differential interference microscopy. Bars, 5 µm.

Actin Oxidation Makes Cells Sensitive to Oxidative Stress, whereas Old Yellow Enzyme Protects Cells from Oxidative Stress
The preceding experiments indicated that Old Yellow Enzyme protects the actin cytoskeleton from the reactive oxygen species (ROS) that result from normal oxidative metabolism. We extended this analysis by addressing the role of actin and Old Yellow Enzyme during extreme oxidative stress. Yeast strains were analyzed for growth in the presence of three reagents commonly used to induce oxidative stress in yeast: H₂O₂, diamide, and menadione. H₂O₂ reacts with metal ions such as Fe²⁺ in the Fenton reaction to produce hydroxyl radicals, whereas the quinone menadione produces a semiquinine radical that is a highly thiol-reactive electrophile that can further react with O₂ to make superoxide anion (Thor et al., 1982). The ROS produced by H₂O₂ or menadione will react with the sulfhydryls of free cysteine residues to initiate disulfide bond formation. Diamide treatment, on the other hand, is not correlated with the production of ROS but reacts directly with thiols to produce disulfides. This can cause oxidative stress in two ways: either indirectly by depletion of reduced glutathione or by directly inducing disulfide bonds within or between proteins (Kosower and Kosower, 1995). For ease of analysis, sensitivity to these agents was determined in halo assays (Figure 7). Cells were mixed at very low densities into a YPD top agar that was poured onto the surface of a YPD plate. Sterile Whatman filter paper discs were placed on the top agar, and dilutions of each agent were spotted onto the Whatman discs and allowed to diffuse outward, allowing us to assess both growth inhibition and viability at various concentrations >1–4 d of growth. At several concentrations of H₂O₂ (Figure 7A), diamide (Figure 7E), and menadione (Figure 7F), the oye2 oye3 strain displayed reduced growth and/or viability relative to the wild-type control strain. Interestingly, this sensitivity was most noticeable for the thiol attacking compound diamide at 100 mM (compare Figure 7E, upper right of each plate) and at 250 mM (compare Figure 7E, lower left of each plate). A strain expressing the actin mutant unable to interact with Oye2p, act1-123 also displayed hypersensitivity to all agents tested but most dramatically to H₂O₂ (Figure 7D, compare 2% H₂O₂ in the upper right corner of each plate). The act1-200 (C374A) and act1-201 (C285A) alleles both suppressed the sensitivity of an Old Yellow Enzyme-deficient strain to all three compounds (H₂O₂ sensitivity is shown in Figure 7C, compare with the right side of Figure 7A), indicating that the sensitivity of the oye strain could be entirely attributed to formation of the actin disulfide bond. Perhaps most surprisingly, the act1-200 and act1-201 alleles confer super-resistance to oxidative stress (compare Figure 7B to A, left plate), suggesting that the actin protein itself is a critically important target that determines cellular sensitivity to oxidative stress.

View larger version (74K): [in this window] [in a new window]   Figure 7. Old Yellow Enzyme and the C285A or C374A actin mutations protect cells from oxidative stress. Strains were plated in top agar on YPD plates upon which Whatman discs were placed and soaked with 10 µl of H2O2 diluted in H2O at (left to right, top to bottom) 1, 2, 5, and 10% (A–D) or 10 µl of diamide diluted in H2O at (left to right, top to bottom) 50, 100, 250, and 500 mM (E) or 10 µl of menadione diluted in H2O at (left to right, top to bottom) 5, 10, 20, and 40 mM (F). Wild-type strain FY86 versus oye2/oye3 strain DAY127 (A, E, and F), act1-200 strain DAY169 versus act1-201 strain DAY171 (B), oye2 oye3 act1-200 strain DAY175 versus oye2 oye3 act1-201 strain DAY173 (C), and wild-type strain TDS167 versus act1-123 strain TDS154 (D) were allowed to grow for 3 d (D and F) or 4 d (A, B, C, and E) at 30°C.

We then examined the actin cytoskeleton in cells recovering from a sublethal dose of H₂O₂. We found that both wild-type and oye2oye3 cells growing in liquid culture could recover from 0.01% H₂O₂ but that the oye cells were significantly delayed (Figure 8A). Approximately 8 h after peroxide addition, the wild-type strain FY86 had resumed growth (as assayed in a Klett meter), whereas the oye strain had not (see vertical line in Figure 8A). Peroxide-treated cultures were fixed at this time point and stained with rhodamine-phalloidin to visualize their actin cytoskeletons. The wild-type strain FY86 (Figure 8B) had a well polarized actin cytoskeleton with cortical patches largely concentrated in the bud and actin cables running from the mother cell body to the bud, a cytoskeletal arrangement consistent with resumption of polarized cell growth. However, in many cases the cortical patches seemed to be aberrantly large and the actin cables excessively long and thick. In contrast, the actin cytoskeleton in the oye strain was not polarized, few if any cables were evident, and most of the F-actin seemed to be focused into large nonpolarized aggregates (Figure 8C).

View larger version (42K): [in this window] [in a new window]   Figure 8. Old Yellow Enzyme is required for recovery of cell growth and actin organization from oxidative stress. Growth of a wild-type strain (FY86) and the oye2 oye3 strain DAY127 was monitored in a Klett Meter after the addition of H2O2 to 0.01% (A). Then, 480 min after the addition of H2O2 to cultures of wild-type strain FY86 (B) and oye2 oye3 strain DAY127 (C), the cells were fixed with formaldehyde, stained with rhodamine-phalloidin, and their actin cytoskeletons visualized by fluorescence microscopy. Bars, 5 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Our results argue that yeast actin can sustain oxidative damage identical to that observed in actin of irreversibly sickled red blood cells. Furthermore, the consequence of actin oxidation is similar in these two very different systems: stabilization of the actin cytoskeleton. Contributions of actin oxidation to actin dynamics would be undetectable in most in vitro approaches because actin is usually (and necessarily) purified in the presence of strong reducing agents. Therefore, this type of regulation could only be uncovered by genetic and cell biological approaches such as those used in this study. The C terminus (including C374) has not been resolved in any of the uncomplexed or unmodified actin structures, indicating that this region of the protein is highly mobile. Results presented here suggest that the C terminus of yeast actin, like red blood cell actin (Bencsath et al., 1996), displays sufficient mobility to adopt conformations that allow C374 to assume proximity to C285. The cytoplasm is a reducing environment and cellular reductants such as reduced glutathione and oxidoreductases such as the thioredoxin and glutaredoxin systems are used to repair inappropriate disulfide bonds in cytoplasmic proteins. Apparently, the actin C285-C374 disulfide is refractory to the action of these systems or its regulation is so important that a specific enzyme, Oye2p, has been recruited by evolution for the task. Interestingly, in expression array experiments OYE2 is strongly induced by H₂O₂, menadione, and diamide treatment and clusters with other oxidative stress response genes such as LYS7, which encodes a copper chaperone for superoxide dismutase, and TRR1, which encodes a thioredoxin reductase (Gasch et al., 2000). It is currently unclear whether the Oye2p oxidoreductase is dedicated to reducing actin or whether it may have other protein disulfide substrates. Our finding that blocking C284-C374 disulfide bond formation in actin can fully suppress the sensitivity to oxidative stress that is caused by the loss of Old Yellow Enzyme suggests that actin is the physiologically most significant substrate of Old Yellow Enzyme. Surprisingly, mutating these cysteines actually makes yeast cells super-resistant to oxidative stress. To our knowledge, this is the first demonstration that the actin cytoskeleton is such an important target of ROS that its oxidation contributes significantly to the inherent sensitivity of cells to oxidative stress. Given the connections between ROS, aging, and apoptosis, it seems likely that actin oxidation may be a contributing factor to these phenomena. For example, because yeast do undergo apoptosis and aging (Laun et al., 2001; Jazwinski, 2002), it will be very interesting to ask whether the C285A and C374A actin mutations prolong yeast cell life or make them refractive to induction of apoptosis. In fact, it was recently shown that reducing actin dynamics increases ROS levels and reduces longevity in yeast cells (Gourley et al., 2004).

An examination of published actin sequences showed that unlike many of the cysteines found in variable positions within actin, C285, and C374 (C284 and C373 in human -actin) are universally coconserved. Given this conservation, C285-C374 disulfide bond formation is likely to occur in many species and isoforms of actin. In fact, actin oxidation may be a contributing factor in many human disease states related to oxidative stress. Determining the contributions of actin oxidation to actin dynamics and function will require sensitive methods for the detection of oxidized actin and biochemical assays of actin dynamics that do not require the presence of strong reducing agents.

Thus far, we have discussed actin oxidation as if it were damage and therefore always having negative consequences to the cell. If this were the case, it is very difficult to understand why both cysteines would be coconserved in all actin species. Furthermore, we have shown that in yeast both of the conserved cysteines of the disulfide pair (C285 and C374) can be mutated with relatively minor consequences. The mutation rate in yeast is very high (1 x 10^-7/base pair/cell division), which combined with selective pressure would be expected to lead to rapid loss of these cysteines if their presence reduced fitness. Therefore, it is reasonable to propose that selective pressure has maintained these cysteines for other reasons, for example, as a mechanism by which cells can control the stability of the actin cytoskeleton. Extending this line of reasoning, it is tempting to theorize that (under certain circumstances) Old Yellow Enzyme may participate in the reverse reaction to catalyze the formation of oxidized actin. The ability to alter the inherent stability of actin filaments either throughout the cell or in specific actin networks would provide the cell with yet another mechanism to regulate actin dynamics and/or to control the cell's energy investment in the actin cytoskeleton. Regardless, our results show that actin oxidation is a common phenomenon in diverse eukaryotic cells. Therefore, it seems likely that Old Yellow Enzyme should be conserved. There are a large number of Old Yellow Enzyme-related NADPH oxido-reductases in the sequence databases and in particular in the human genome. Because this is such a large enzyme family, identification of a true Oye2p homologue is likely to require functional assays such as complementation of a yeast oye strain.

In summary, the work presented here makes four important contributions. After many years of investigation, we can now assign at least one physiologically important function for Old Yellow Enzyme and a probable cellular substrate. Second, it seems that actin oxidation contributes to actin dynamics in diverse systems and may in fact be a mechanism for cells to regulate actin cytoskeleton stability. Third, we have shown that the yeast oye knockout strain can serve as a model for studying human diseases, such as sickle cell crisis, in which actin oxidation is a contributing factor. Finally, we have shown that formation of a specific disulfide bond in actin is a large contributor to the cellular sensitivity to oxidative stress.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank D. Botstein for advice and convincing us "that the Old Yellow Enzyme–actin two-hybrid interaction had to be important"; the late V. Massey and B.-J. Brown for encouragement, providing OYE protein, and welcoming us into the OYE field; R. Cross and P.M. Kane for advice and critical reading of the manuscript; S. Loh for advice and technical assistance; and T. Duncan for advice, technical assistance, and help with the molecular rendering. This research was supported by National Institutes of Health grant GM-56189. This article is dedicated to the memory of Vincent Massey whose life work was Old Yellow Enzyme.

	Footnotes

 
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E04–06–0445. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–06–0445.

Abbreviations used: AD, activation domain; DBD, DNA binding domain; FMN, flavin mononucleotide; ISC, irreversibly sickled red blood cell; OYE, Old Yellow Enzyme; ROS, reactive oxygen species; RSC, reversibly sickled red blood cell.

^* Corresponding author. E-mail address: ambergd{at}upstate.edu.

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