From the Dipartimento di Chimica Biologica and
§ Dipartimento di Biologia, viale G. Colombo, 3, 35121 Padova, Italy and ¶ Molecular and Cell Biology, Institute of
Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, United Kingdom
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ABSTRACT |
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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 (1-3). 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 single-domain 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 heme-dependent 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.
Strains, Vectors, and Media
Escherichia coli strains INV S. cerevisiae strains W303-1B/A (MATa, ade2-1,
his3-11, 15, leu2-3, 112, trp1-1, ura3-1, can1-100) (14),
W3/H (MATa, hem1::HIS3) (15) and FY73
(MAT Cosmid 14-5 was provided by P. Philippsen (University of Basel). This
cosmid contains a 38.8-kilobase pair DNA fragment from chromosome XIV
of the S. cerevisiae strain FY1679.
Analysis of Yeast Transcripts
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 × 106 cells/ml. Then half of the cells were allowed to grow
aerobically to a density of 2 × 107 cells/ml and
subsequently harvested for analysis (AIR), and the other half was
transferred to an air-tight flask as described in Ohaniance et
al. (17) and shifted to anaerobic conditions by vigorous bubbling
of ultrapure N2 for 30 min. The latter culture was shaken
under N2 pressure until it reached a density of 2 × 107 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 Heme Depletion (Fig. 3B)--
Cells of wild type
W303-1B/A and of W3/H mutant (hem1) were grown in YPGal
medium supplemented with 0.18 mg/l 5-aminolevulinic acid (Sigma) at
28 °C to a density of 2 × 107 cells/ml.
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
A600 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 µg/ml uracil, 20 µg/ml histidine)
until the A600 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 µg/ml uracil, 20 µg/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 A600 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
Na2HPO4, 4 mM EDTA, 1% SDS, 50% formamide, pH 7.2 (Fig. 3, A and B) or at
65 °C in 0.5 M Na2HPO4, 7% SDS,
1 mM EDTA, pH 7.2 (Fig. 3C). Filters were washed
in 2× SSC (0.3 M NaCl, 0.03 M sodium citrate,
pH 7.0) for 15 min at 65 °C and then in 2× SSC containing 0.1% SDS
for 15 min at 65 °C. DNA fragments of the YNL234w and
ACT1 genes were 32P-labeled to specific
activities greater than 5 × 108 dpm/µg of DNA by
random-priming, whereas the end-labeled oligonucleotide 5'-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 stationary-phase 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 3' end has been
amplified by PCR from DNA of cosmid 14-5 using primers A
(5'-GCTGGTTGCATATGACAGGA-3') and B (5'-ATTACTTTCGTCGACGGTAC-3'). 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(+) (Novagen). The
resulting plasmid, pYNL234-His6 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 µg/ml ampicillin and 10 mM 5-aminolevulinic acid at 37 °C
until an A600 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
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
INTRODUCTION
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Abstract
Introduction
References
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Fig. 1.
Structural organization of some hemoglobins
from yeast and bacteria. A, the single domain protein
of Vitreoscilla hemoglobin (Vit). B,
two examples of bifunctional hemoglobins: the S. cerevisiae
flavohemoglobin (Sac) and the R. meliloti FixL
protein (FixL); in this group FixL represents an exception,
as the globinic domain is preceded by four hydrophobic transmembrane
regions (black boxes) that anchor the protein to the
membrane (adapted from Monson et al. (4)). C, the
unique structural organization of the Ynl234wp protein (Ynl234wp). The
light gray boxes represent the globin domains.
aa, amino acids.
MATERIALS AND METHODS
F' (Invitrogen), used
for amplification of the plasmids, and BL21 (DE3) (hsdS,
gal [
cIts857 ind1
Sam7 nin5 lacUV5-T7
gene1]), used for expression and purification of the
recombinant protein, were grown in Luria-Bertani (LB) medium (1% Difco
bacto tryptone, 0.5% Difco yeast extract, 0.5% NaCl).
, his3
200, ura3-52) (16) were used for RNA preparations, and strain FY1679
(MATa/MAT
, ura3-52/ura3-52, his3
200/HIS3, leu2
1/LEU2, trp1
63/TRP1) (16) was used for the gene disruption experiments.
-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.
RESULTS AND DISCUSSION
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.
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Fig. 2.
Amino acid sequence alignment of the Ynl234wp
central domain (from amino acids 164 to 301) with known yeast and
bacterial Hb domains. The multiple alignment has been performed
using the PILEUP program of the GCG Software Package (University of
Wisconsin). The topological regions (A to H),
marked on the top of the sequences, were identified according to the
templates of Moens et al. (27). The triangles
indicate the positions of residues considered to be important for heme
binding or in ligand recognition, whereas the circles
indicate the only two residues absolutely invariant in all known
globins, the proximal histidine (F8) and the CD1 phenylalanine.
Sac, S. cerevisiae flavohemoglobin (PIR accession
number A45383); Vit, Vitreoscilla hemoglobin (PIR
accession number AO2564); Alc, Alcaligenes
flavohemoprotein (GenBankTM accession number X74334);
Hmp, E. coli flavohemoprotein (PIR accession
number S15992); Can, Candida flavohemoglobin
(GenBankTM accession number X68849); Ynl,
YNL234w gene product (SwissProt accession number
P53857).
The remaining NH2- 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 (33-35). The induction of YNL234w transcription by several stress
conditions is in agreement with the presence of two STRE sequences at
positions 68 (5'-CCCCT) and
182 (5'-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.
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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 Program2). 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(+) 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
Ni2+ 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 heme-associated 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).
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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 hemoprotein, 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 (41-43)
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).
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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.
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ACKNOWLEDGEMENTS |
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We thank Jacqueline Verdière for her suggestions and for kindly providing the heme-depleted strain and Luc Moens for making for us an independent alignment of the Ynl234wp central domain, which confirmed the results shown in Fig. 2. We are very grateful to Paolo Ascenzi for helpful discussions and critical reading of the manuscript.
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FOOTNOTES |
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* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a grant from European Community as part of the
EUROFAN Program.
** To whom correspondence should be addressed. Tel.: +39 +049 8276141; Fax: +39 +049 8073310; E-mail: carigna{at}civ.bio.unipd.it.
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ABBREVIATIONS |
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The abbreviations used are: Hb, hemoglobin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
2 G. Sartori, G. Mazzotta, S. Stocchetto, A. Pavanello, and G. Carignani, manuscript in preparation.
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REFERENCES |
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