Characterization of a New Hemoprotein in the Yeast Saccharomyces cerevisiae *

The Saccharomyces cerevisiae gene YNL234w encodes a 426-amino acid-long protein that shares significant similar-ities with the globin family. Compared with known globins from unicellular organisms, the Ynl234wp polypeptide is characterized by an unusual structure. In this protein, a central putative heme-binding domain of about 140 amino acids is flanked by two sequences of about 160 and 120 amino acids, respectively, which share no similarity with known polypeptides. Northern analysis indicates that YNL234w transcription is very low in cells grown under normal aerobic conditions but is induced by oxygen-limited growth conditions and by other stress conditions such as glucoserepression,heatshock,osmoticstress,andnitrogenstarvation.However,thedeletionofthegenehadnodetect-ableeffectonyeastgrowth.TheYnl234wppolypeptidehasbeenexpressedin Escherichia coli , and the hemoprotein nature of the recombinant protein was demonstrated by heme staining after SDS/polyacrylamide gel electrophoresis and spectroscopic analysis. Our data indicate that purified recombinant Ynl234wp possesses a noncovalently bound heme molecule that is predominantly found in a low spin form. Spectrophotometric Assays Spectrophotometric measurements were made using a Perkin-Elmer Lambda 5 UV/VIS spectrophotometer at room temperature. Absolute spectra were recorded by scanning the purified protein preparation against the protein buffer (20 m M Tris-HCl, 0.3 M NaCl, 10% glycerol, pH 7.5) before and after the addition either of an excess of potassium ferricyanide or of few grains of sodium dithionite. The CO difference and absolute spectra were obtained by bubbling CO directly in the cuvette for a few seconds immediately before measurements (26).

The Saccharomyces cerevisiae gene YNL234w encodes a 426-amino acid-long protein that shares significant similarities with the globin family. Compared with known globins from unicellular organisms, the Ynl234wp polypeptide is characterized by an unusual structure. In this protein, a central putative heme-binding domain of about 140 amino acids is flanked by two sequences of about 160 and 120 amino acids, respectively, which share no similarity with known polypeptides. Northern analysis indicates that YNL234w transcription is very low in cells grown under normal aerobic conditions but is induced by oxygen-limited growth conditions and by other stress conditions such as glucose repression, heat shock, osmotic stress, and nitrogen starvation. However, the deletion of the gene had no detectable effect on yeast growth. The Ynl234wp polypeptide has been expressed in Escherichia coli, and the hemoprotein nature of the recombinant protein was demonstrated by heme staining after SDS/polyacrylamide gel electrophoresis and spectroscopic analysis. Our data indicate that purified recombinant Ynl234wp possesses a noncovalently bound heme molecule that is predominantly found in a low spin form.
Hemoglobins (Hbs) 1 are widely distributed in many organisms, including higher plants, protozoa, fungi, and bacteria (1). In contrast to their vertebrate counterparts, the nonvertebrate globins exhibit an extensive variability of their structural organization (1)(2)(3). In bacteria and yeasts, Hbs are found as bifunctional proteins in which the N-terminal globin domain is linked to a second domain with a different activity (see Fig. 1). The C-terminal region can be either a FAD/NAD(P)H-binding domain with oxidoreductase activity (flavohemoglobins) or a kinase domain, as in the nitrogen-fixing bacterium Rhizobium meliloti (5). The heme-binding domains of the flavohemoglobins share substantial sequence similarity with the singledomain Hb of the purple bacterium Vitreoscilla, which works in association with an NADPH reductase encoded by a different gene (6). Although the function of these proteins is still unclear, oxygen appears to play a role in the expression of at least some of them. For example, growth in limiting concentrations of oxygen induces the synthesis of the Vitreoscilla Hb (7) and of the Bacillus subtilis (8) and Alcaligenes eutrophus (9) flavohemoglobins. In contrast, transcription of the Saccharomyces cerevisiae flavohemoglobin, encoded by the YHB1 gene, is enhanced under oxygen-replete conditions via the hemedependent activation of the HAP1 and HAP2/3/4 transcription factors (10). Recently, it has been proposed that this flavohemoglobin might play a role in the oxidative stress response in yeast (11), but discrepancies in the results between different laboratories do not allow this conclusion (12); however, the results of phenotypic studies on YHB1-deleted or overexpressing strains suggest a complex relationship between Yhb1p function and cell defense against various stresses (12).
Here we report the study of a gene located on chromosome XIV of S. cerevisiae that encodes a new putative hemoglobin, showing peculiar structural characteristics in comparison with known hemoglobins. Gene YNL234w was identified during the sequencing of the entire S. cerevisiae genome (13). It encodes a 426-amino acid-long polypeptide that is characterized by the presence of a central domain of about 140 amino acids, showing a significant similarity to the globin family. This domain is flanked by two sequences of about 160 and 120 amino acids, respectively, that share no similarity with known protein domains or motifs (see Fig. 1). We show here that a recombinant, heterologously expressed Ynl234wp polypeptide actually binds heme and that expression of the YNL234w gene in yeast is induced by hypoxia and other stress conditions.

Growth Conditions
Effect of Hypoxia (see Fig. 3A)-Cells of strain W303-1B/A were grown aerobically at 28°C in YPGal medium (1% yeast extract, 1% bactopeptone, 2% galactose, 20 mg/ml adenine) supplemented with 0.2% Tween 80 and 30 mg/l ergosterol (Sigma) up to a density of 2 ϫ 10 6 cells/ml. Then half of the cells were allowed to grow aerobically to a density of 2 ϫ 10 7 cells/ml and subsequently harvested for analysis (AIR), and the other half was transferred to an air-tight flask as de-* This work was supported by MURST-Università di Padova Cofin 1997. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ Supported by a grant from European Community as part of the EUROFAN Program.
** To whom correspondence should be addressed. Tel.: ϩ39 ϩ049 8276141; Fax: ϩ39 ϩ049 8073310; E-mail: carigna@civ.bio.unipd.it. 1 The abbreviations used are: Hb, hemoglobin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis. scribed in Ohaniance et al. (17) and shifted to anaerobic conditions by vigorous bubbling of ultrapure N 2 for 30 min. The latter culture was shaken under N 2 pressure until it reached a density of 2 ϫ 10 7 cells/ml; then, before opening, the flasks were transferred to ice and chilled for 30 min, and the cells were harvested for analysis (N2).
Effect of Glucose Concentration-Cells of strain FY73 were grown at 30°C in YP medium (1% yeast extract, 1% bactopeptone) supplemented with 2% glycerol and 1% ethanol until the A 600 reached 0.8, whereupon one-third of the cells were harvested for analysis (Fig. 3C, lane 1). The remaining cells were washed in sterile distilled water, resuspended in an equal volume of YP containing 2% glucose (YPD), and grown for 60 min at 30°C. Half of these cells were harvested for analysis (Fig. 3C, lane 2), whereas the remaining cells were harvested after a further 24 h of growth (Fig. 3C, lane 3).
Nitrogen Starvation and Osmostress-Cells of strain FY73 were grown at 30°C in GYNB (0.67% yeast nitrogen base without amino acids, 4% glucose, 20 g/ml uracil, 20 g/ml histidine) until the A 600 reached 0.8. One-third of these cells was harvested for analysis (control at 30°C; Fig. 3C, lane 4). The second third of these cells was washed in sterile distilled water, resuspended in two volumes of GYNB without ammonium (0.67% yeast nitrogen base without amino acids and ammonium sulfate, 4% glucose, 20 g/ml uracil, 20 g/ml histidine), and starved for 2 h at 30°C (nitrogen starvation; Fig. 3C, lane 5). NaCl was added (0.7 M final concentration) to the final third of cells, which were harvested after 60 min at 30°C (osmostress; Fig. 3C, lane 6).
Heat Shock-Cells of strain FY73 were grown at 23°C in GYNB to an A 600 of 0.8, whereupon half of the cells were taken for analysis (control at 23°C; Fig. 3C, lane 7). The other half was incubated at 36°C for 30 min before harvesting (heat shock; lane 8).

Northern Analysis
Total RNA was prepared by a phenol extraction procedure as described previously (18) and subjected to electrophoresis in a formaldehyde-denaturing agarose gel as described in Sambrook et al. (19). Northern blot hybridization was performed either at 42°C in 0.7 M NaCl, 0.05 M Na 2 HPO 4 , 4 mM EDTA, 1% SDS, 50% formamide, pH 7.2 ( Fig. 3, A and B) or at 65°C in 0.5 M Na 2 HPO 4 , 7% SDS, 1 mM EDTA, pH 7.2 (Fig. 3C). Filters were washed in 2ϫ SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) for 15 min at 65°C and then in 2ϫ SSC containing 0.1% SDS for 15 min at 65°C. DNA fragments of the YNL234w and ACT1 genes were 32 P-labeled to specific activities greater than 5 ϫ 10 8 dpm/g of DNA by random-priming, whereas the end-labeled oligonucleotide 5Ј-CCTCCGCTTATTGATATGCTTAAG (20) was used to probe the 25 S rRNA gene. Hybridization signals were quantified by direct two-dimensional phosphorimaging of Northern membranes using the Bio-Rad GS-525 Molecular Imager ® system.

YNL234w Gene Disruption and Phenotypic Tests
The YNL234w gene disruption was performed by the PCR-based method of Wach et al. (21). The diploid yeast strain FY1679 was transformed by the lithium acetate procedure (22) with a PCR-amplified DNA fragment containing the kanMX4 module (conferring resistance to geneticin) of plasmid pFA6-kanMX4 (21) flanked by two 40-base pair S. cerevisiae sequences corresponding to regions located immediately upstream and downstream of the YNL234w coding sequence. Haploid ynl234w mutants were obtained by sporulation of heterozygous diploids, and selection of spores was performed on geneticin-containing medium. The correct chromosomal insertion of the kanMX4 module was verified by PCR amplification using primers specific for the disruption cassette and the chromosome XIV DNA regions flanking the YNL234w gene. 2 For the phenotypic analysis, several dilutions of fresh stationaryphase cultures of mutant strains were spotted together with the corresponding wild-type strains on YP plates (1% yeast extract, 1% bactopeptone) containing different carbon sources (2% glucose, 2% galactose, 3% glycerol). The growth was followed for 5 days at three different temperatures (16, 28, and 36°C). For the heat shock experiments, cells were grown in YPD medium at 25°C until exponential phase, when half of the cells were exposed to 38°C for 20 min in a water bath, whereas the remaining cells were left at 25°C. Cells were then diluted, plated on YPD, and incubated at 25°C for 5 days, when sizes of the colonies and percentage of survivors were determined.

Cloning, Expression, and Purification of Recombinant Ynl234wp
A DNA fragment corresponding to the YNL234w coding sequence lacking the 87 last nucleotides at the 3Ј end has been amplified by PCR from DNA of cosmid 14 -5 using primers A (5Ј-GCTGGTTGCATATGA-CAGGA-3Ј) and B (5Ј-ATTACTTTCGTCGACGGTAC-3Ј). These introduce, respectively, an NdeI site by the ATG codon of the gene and a SalI site 29 codons upstream of the stop codon. DNA amplification was performed using a Pfu (Stratagene) and Taq (Life Technologies, Inc.) DNA polymerase mix in a 1:5 ratio. The product of amplification was digested with NdeI and SalI restriction enzymes and cloned into the expression vector pET-20b(ϩ) (Novagen). The resulting plasmid, pYNL234-His 6 allows the synthesis in a bacterial T7 expression system (23) of a recombinant 46-kDa Ynl234wp protein with a C-terminal tag of six histidines. The sequence of the cloned fragment was verified by automated dideoxy sequencing (ABI373 DNA sequencer, Applied Biosystem).
Cells of the E. coli strain BL21(DE3), transformed with the recombinant plasmid, were grown in LB medium containing 100 g/ml ampicillin and 10 mM 5-aminolevulinic acid at 37°C until an A 600 nm of 0.5-0.6, when the temperature was shifted to 20°C and transcription of the YNL234w coding sequence was induced by addition of 0.4 mM isopropyl ␤-D-thiogalactopyranoside. Induction at the usual 37°C temperature resulted in higher yields but also in a prevalent insoluble form of the protein. After 5-7 h of further incubation, cells were harvested and resuspended in 10 ml of SB (20 mM Tris-HCl, 0.3 M NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, pH 8.0)/g of wet pellet and sonicated. Purification of the recombinant protein was performed according to the manufacturer's protocol by an affinity column containing nickel nitrilotriacetic acid-agarose (Quiagen). Recombinant YNL234wp was eluted with 10 ml of SB containing 100 mM imidazole. Orange-colored fractions were collected and dialyzed against SB (without phenylmethylsulfonyl fluoride) at pH 7.5. The purity of the preparation was assessed by SDS/PAGE according to Laemmli (24), and protein concentration was determined by the Bradford method (Bio-Rad) using bovine serum albumin as a standard. For N-terminal sequencing, the recombinant polypeptide was electroblotted after SDS-PAGE to the PVDF membrane (ProBlott, Applied Biosystems), visualized with Coomassie Blue, and analyzed by automated Edman degradation.

Heme Staining
Heme staining of purified Ynl234wp recombinant protein was carried out with tetramethylbenzidine after SDS-PAGE was performed without the addition of sulfhydryl reducing agents according to Thomas et al. (25). The proteins were loaded in different amounts because of the lower sensitivity of the staining method for human Hb.

Spectrophotometric Assays
Spectrophotometric measurements were made using a Perkin-Elmer Lambda 5 UV/VIS spectrophotometer at room temperature. Absolute spectra were recorded by scanning the purified protein preparation against the protein buffer (20 mM Tris-HCl, 0.3 M NaCl, 10% glycerol, pH 7.5) before and after the addition either of an excess of potassium ferricyanide or of few grains of sodium dithionite. The CO difference and absolute spectra were obtained by bubbling CO directly in the cuvette for a few seconds immediately before measurements (26).

The Central Region of the Ynl234wp Polypeptide Shows the Characteristics of a Hemoglobin Domain
The 426-amino acid-long sequence of the polypeptide encoded by the YNL234w gene was compared with protein data bases, and a weak but significant similarity was found with several hemoglobin chains (13). In fact, the amino acid sequence of the Ynl234wp central region matches fairly well to the amino acid sequence template established by Moens et al. (27) based on the alignment of nonvertebrate hemoglobin sequences. It matches also, but with a higher penalty score, to the template based on vertebrate sequences established by Bashford et al. (28). The alignment of the central part of the Ynl234wp polypeptide (amino acids 164 -301) with five hemoglobin domains from unicellular organisms is shown in Fig. 2. The ␣ helical motifs (A-H) known to compose the globin structure around the heme group have been identified on the basis of the nonvertebrate template (27). Residues known to be important in establishing contacts with heme or in controlling the ligand binding are indicated (for a review, see Bolognesi et al. (29)). These comparisons identify the central part of the Ynl234wp polypeptide as a putative heme-binding domain.
The remaining NH 2 -and C-terminal regions (163 and 125 amino acids, respectively) do not show any significant homology to known proteins; nevertheless these regions might represent functionally distinct domains. The overall structural organization of Ynl234wp makes this protein unique among known hemoglobins from vertebrate or nonvertebrate organisms (see Fig. 1).

Transcription of the YNL234w Gene Is Induced by Hypoxia and Other Stress Conditions
Expression of the YNL234w gene in yeast cells was shown by Northern blot analysis. A weak band of about 1.4 kilobases, which is in agreement with the size of the gene, indicates that the YNL234w gene is expressed at low levels in cells grown under normal aerobic conditions. Transcription of the gene, however, is significantly enhanced in cells grown under hypoxia (Fig. 3A) and under different stress conditions as glucose repression, nitrogen starvation, osmostress, and heat shock ( Fig. 3C and Ref. 30). These data identify S. cerevisiae YNL234w as a hypoxic and a stress-responsive gene. Hypoxic gene expression in yeast is regulated by two different systems. The first involves the heme molecule and, in the majority of the cases, the transcriptional regulators Hap1p and Rox1p; the second is heme-independent, but little is known about its mechanism (31,32). Preliminary results indicate that expression of the YNL234w gene is moderately increased in heme-depleted cells in comparison to wild-type cells (Fig. 3B). However, as the increase of YNL234w transcription in the case of the absence of heme is moderate (about 3-fold) in comparison with the one observed in the case of hypoxia (about 20-fold), it is conceivable that regulation of YNL234w transcription by oxygen is mediated both by the heme molecule and by a heme-independent mechanism. The mechanism of the general stress response in yeast has not been fully elucidated; however, it is known that, in response to diverse stress conditions, two homologous zinc finger proteins, Msn2p/Msn4p, activate gene transcription by binding to the stress response element STRE (33)(34)(35). The induction of YNL234w transcription by several stress conditions is in agreement with the presence of two STRE sequences at positions Ϫ68 (5Ј-CCCCT) and Ϫ182 (5Ј-AGGGG) from the first ATG codon. Examples of yeast genes whose transcription is controlled both by oxygen and by different stress conditions are the iso-2-cytochrome c gene CYC7 (36,37) and the catalase gene CTT1 (33). Expression of the ROX3 gene, which encodes an essential protein that contributes to the global stress response (37,38), is also induced by oxygen starvation and several stress conditions; however, the effect of oxygen is not mediated by heme, and the stress response is not activated through the STRE sequence. It has recently been shown that Rox3p is a component of the multiprotein complex "mediator," which is part of the RNA polymerase II holoenzyme (39). We believe that a better understanding of the mechanisms that control YNL234w expression, and in particular, of the relationships existing between oxygen starvation and other stress conditions in inducing its transcription could give some interesting insights into the function of this new putative hemoglobin in the yeast cell.

The YNL234w Gene Is Not Essential for Yeast Growth
The YNL234w gene was deleted in the yeast strain FY1679 using the PCR-based technique described by Wach et al. (21) (this work was performed under the Eurofan I Biotech Program 2 ). A preliminary analysis of the disrupted haploid strains was performed by growing them at different temperatures on solid media containing different carbon sources, but no significant phenotypic differences between the wild-type and the mutant strains were observed. The sensitivity of the mutant strains to heat shock treatment was tested by shifting exponentially growing cultures from 25 to 38°C and then determining the percentage of survivors on YPD plates, but also in this case, a clear difference between the wild-type and the mutant strains was not observed. However, if the role of the YNL234w gene, as suggested by the transcription data, is to mitigate cellular damage in different stress conditions, more accurate analysis seems to be required to highlight a phenotype of the YNL234w-deleted strains.

Recombinant Ynl234wp Binds Noncovalently a Low Spin Form Heme Molecule Expression and Purification of Recombinant Ynl234wp Protein
To facilitate the characterization of the Ynl234wp protein, which is normally poorly expressed in yeast, we decided to purify a heterologously expressed protein. A PCR-fragment corresponding to the nucleotide sequence encoding a Ynl234wp polypeptide lacking the 29 C-terminal residues was obtained by amplification of the genomic DNA of S. cerevisiae strain FY1679. The fragment was cloned in the isopropyl ␤-D-thiogalactopyranoside-inducible pET-20b(ϩ) E. coli expression vector, upstream of and in-frame with a short plasmidic sequence coding for eight amino acids (DKLAAALE) followed by six residues of histidine (His-tag). Nucleotide sequencing of the cloned fragment confirmed its identity with the wild-type sequence.
E. coli cells transformed with this plasmid were grown in liquid culture supplemented with the heme precursor 5-aminolevulinic acid to increase de novo heme synthesis (40). After induction with isopropyl ␤-D-thiogalactopyranoside, a prominent protein band of the expected 46 kDa was visible in bacterial extracts. This was purified to near homogeneity by a single-step affinity chromatography utilizing the chelating ligand nitrilotriacetic acid charged with Ni 2ϩ ions. The identity of the 46-kDa band present in the orange-colored fractions with the recombinant Ynl234wp polypeptide was confirmed by N-terminal sequencing, which revealed the expected TGEKI sequence.

Biochemical Characterization of the Ynl234wp Recombinant Protein
Heme Staining-The purified recombinant Ynl234wp protein was analyzed by benzidine heme staining after SDS-PAGE (25). This method allows detection of very low levels of hemeassociated peroxidase activity. Furthermore, the denaturing conditions of electrophoresis led to a loss of heme from hemoproteins in which the prosthetic group is noncovalently bound and the free heme appears as a diffuse band with a mobility similar to that of the bromphenol blue dye. By this simple method, it is possible to distinguish between hemoproteins with covalently and noncovalently bound heme. Intense staining of Ynl234wp and the presence in the same electrophoretic lane of abundant free heme clearly indicate that this protein does bind heme and that the prosthetic group is noncovalently bound (Fig. 4).
Spectroscopic Analysis-The absolute visible spectrum of purified recombinant Ynl234wp protein at pH 7.5 is shown in Fig.  5A, trace 1. The spectrum shows the characteristics of a hemo- protein, with an intense Soret band positioned at 411 nm, but the ␣ and ␤ peaks are not well resolved. This profile is not modified by the addition of potassium ferricyanide (data not shown), indicating that the hemoprotein is already in its oxidized form. After the addition of sodium dithionite, the optical spectrum is characterized by a Soret peak shifted to 426 nm, a minor ␤ peak centered at 530 nm, and a major ␣ peak at 559 nm (Fig. 5A, trace 2). These spectroscopic profiles are characteristic of hemichromes (41)(42)(43) and suggest that the major part of the protein, probably obtained in the form of high spin met form, has been converted to low spin hemichromes. This hypothesis is confirmed by the CO difference spectrum (Fig.  5B, trace 3) in which the trough present at the same wavelength of the ␣ peak (559 nm) has the characteristics of low spin heme molecules; however, the intensity of the trough is not as pronounced as those observed for true low spin hemoproteins (43), suggesting that the heme molecule bound to Ynl234wp may be present as a mixture of low and high spin states, as already observed for the heterologously expressed Hb of the unicellular alga Clamydomonas eugametos (44) and for the Hb from the cyanobacterium Nostoc commune (45). The susceptibility to autoxidation and hemichrome formation varies with different Hbs, and the rates of the two processes are relatively rapid for some Hbs, as is the case of the myoglobin isolated from Paramecium caudatum (46).
The knowledge of the real redox state of the heme molecule bound to Ynl234wp, together with the role played by the two regions flanking the heme-binding domain, will be necessary elements to understand the function of this protein in the yeast cell. The results presented here arouse the interest in this new hemoprotein of S. cerevisiae, whose expression is induced in cells grown under hypoxia and other stresses and that is probably necessary to the cell under these severe conditions. The two regions flanking the heme-binding domain, which have no apparent homologs in the yeast cell, might actually represent new functions or, more likely, be docking domains responsible for the recruitment of proteins whose activation (or inactivation) is necessary to the cell grown under these particular conditions. FIG. 5. A, absolute spectra of the purified Ynl234wp protein (trace 1) and of the dithionite-reduced form (trace 2) at pH 7.5. Trace 3 represents the analogous spectrum for the fraction of control E. coli cells. In the upper panel, a 4ϫ magnification of the 500 -600-nm region is shown. B, absolute spectra of the CO complex (trace 1) and of the dithionite-reduced form (trace 2) of Ynl234wp. Trace 3 represents the CO difference spectrum (reduced ϩ CO Ϫ reduced). In the 4ϫ magnification of the 500 -600-nm region of trace 3, the straight line has been used to determine the characteristics of the 559-nm trough by the criteria of Wood (43).