(Received for publication, November 1, 1994; and in revised form, December 27, 1994)
From the
The Saccharomyces cerevisiae hemoglobin is a flavoprotein of unknown function. It shares extensive sequence homology with the globin of Candida as well as those of several bacterial species. We have studied its gene regulation in order to better understand its purpose in the cell. Transcriptional analyses indicate that, in sharp contrast to the bacterial globins of Vitreoscilla and Alcaligenes eutrophus, the S. cerevisiae globin message is induced during logarithmic growth and under oxygen-replete conditions. Transcription of the S. cerevisiae hemoglobin gene is positively regulated by the transcription factors heme-activated protein (HAP) 1 and HAP2/3/4, which respond to intracellular heme levels. Anaerobically, there is a low level, HAP-independent induction of hemoglobin mRNA. Unlike other systems influenced by the HAP2/3/4 transcription factor complex, no activation of hemoglobin expression by growth in non-fermentable carbon sources is observed. Flavohemoglobin gene disruption does not alter cell viability or growth in a variety of oxygen conditions and carbon sources. Physical and genetic mapping of the S. cerevisiae flavohemoglobin gene places it on chromosome seven near the formyltetrahydrofolate synthase (ADE3) locus. These data indicate that, despite the high degree of homology, the S. cerevisiae globin may have a function distinct from those proposed for bacterial globins.
Hemoglobins have been detected spectrally and their genes cloned
from organisms representing all kingdoms of life, indicating a
primordial origin and a continuing widespread requirement for this
protein(1, 2, 3) . Although the role of
vertebrate hemoglobins as facilitators of oxygen diffusion is well
established, the function of globins in invertebrate animals, as well
as in plants, protozoa, fungi, and bacteria, is generally unclear.
Clues to the function of the microorganismal globins have come through
heterologous expression of hemoglobin from the aerobic bacteria Vitreoscilla. Overproduction of the Vitreoscilla globin (VtHb) ()allows increased cell densities and
rescues terminal oxidase mutants in Escherichia coli,
indicating that VtHb is capable not only of delivering molecular oxygen
but also of facilitating its reduction in
vivo(4, 5) . Hemoglobins recently sequenced from
the bacteria E. coli and Alcaligenes eutrophus and
the yeasts Saccharomyces cerevisiae and Candida
norvegensis are two-domain proteins with nearly 40% sequence
identity(6, 7, 8, 9) . Their
N-terminal regions share substantial sequence homology with the
single-domain VtHb, whereas the C terminus contains a reductase domain
with potential binding sites for flavin (FAD) and NADPH. Because of the
additional domain, this class of globins has also been called
flavohemoglobins. The native VtHb interacts with a separate reductase,
making it a two-polypeptide analog of the
flavohemoglobins(10) .
Gene regulation studies of the bacterial globins suggest a correlation in the functions of these proteins. Hemoglobin levels increase over 50-fold in Vitreoscilla as cell density increases and oxygen tension drops(11) . There is also an increase in reductase levels under the same conditions, suggesting coordinate expression of the hemoglobin and reductase activities in Vitreoscilla(10) . Remarkably, the oxygen regulation of Vitreoscilla hemoglobin can also be seen using the native VtHb promoter expressed in E. coli(12, 13) . Transcriptional fusions to the hemoglobin promoter show a 5-7-fold increase in reporter gene expression when cells are shifted to microaerophilic conditions or when cells reach the end of log-phase growth. Limited oxygen supply also causes a 20-fold increase in A. eutrophus globin levels(14) . On the basis of promoter sequence homology, the FNR and NARL transcription factors, which control anaerobic gene expression in E. coli, have been proposed to affect expression of the A. eutrophus globin(9) . A putative binding site for FNR is also found in the promoter of the Vitreoscilla gene, and E. coli mutants lacking the FNR gene product do not show microaerophilic activation of heterologous VtHb transcription(15) .
In S. cerevisiae, transcription factors involved in oxygen regulation of gene expression have been well characterized(16, 17, 18) . Heme, the biosynthesis of which requires molecular oxygen in eukaryotes (19) , is an effector molecule involved in the sensing of intracellular oxygen levels. For aerobically induced genes, HAP1 and the HAP2/3/4 complex activate transcription in the presence of heme(16) . Heme can also inhibit the transcription of hypoxic or anaerobically expressed genes through the ROX1 and ORD1 transcription factors(20, 21) . To investigate possible functions for the S. cerevisiae flavohemoglobin, we have initiated gene regulation and disruption studies. Our results indicate that S. cerevisiae hemoglobin expression is regulated by cell density and oxygen in a manner different from that of the bacterial globins, suggesting a separate and as yet undefined role for this protein in aerobic yeast metabolism.
The yeast hemoglobin
reporter gene construct (pYH10) was generated by subcloning a SpeI-NheI restriction fragment containing 1.6 kb
of YHG promoter into the SpeI site of the lacZ expression plasmid yIP357R(23) . Standard techniques in
the manipulation of these shuttle vectors and propagation of E.
coli were used(24) . Yeast transformations were performed
using the LiAc method as described (25) and plated onto
selective media(26) .
Figure 1:
Repression of YHG mRNA with increasing
cell density. Shown is the growth of strain YM4134 as a function of
time after an initial inoculation of 10 cells. Cultures
were grown in YPGal media with vigorous shaking at 30 °C. At the
points indicated by the arrows, aliquots of culture were
removed and total RNA isolated. The numbers above the inset indicate the A
at the time of RNA
isolation. 10 µg of total RNA was loaded into each lane.
The RNA blot was probed with a radiolabeled YHG PCR product as
described under ``Materials and Methods.'' Densitometry
readings of the band intensities (setting A
of
1.5 equal to 1.0) were, in order: 1.0, 1.0, 0.78, 0.69, 0.04, <0.01,
and <0.01. Ethidium-stained intensity of the 18 S rRNA band was used
as an internal RNA loading standard and gave intensities: 1.0, 1.0,
1.0, 1.2, 1.2, 0.93, and 0.81.
Figure 2: YHG gene expression at varied oxygen tensions. YM4134 was grown in YPGal at the defined oxygen concentrations shown above the figure. Oxygen tensions were maintained by bubbling defined gas mixtures continuously through the cultures. All cultures also contained 10 µg/ml ergosterol and 0.1% Tween 80 to allow anaerobic growth. After midlog phase was reached, cells were chilled on ice with continued bubbling for 20 min, and RNA was isolated. The blot was hybridized with a mixture of YHG and TIF51B probes prepared as described under ``Materials and Methods.'' TIF51A is the 90% identical aerobic gene partner of TIF51B, which cross-hybridizes to TIF51B probe. This blot was stripped and re-hybridized with a S. cerevisiae ACT1 probe to ensure equal loading of mRNA. Densitometry readings of the YHG message gave the following intensities (setting 21% as 1.0): 1.26, 1.0, 0.50, 0.28, 0.23, 0.32, and 0.23.
Given the oxygen
responsiveness of the YHG gene, we examined YHG mRNA levels in a S.
cerevisiae strain (nem1) deleted in -aminolevulinic
acid synthase, the enzyme catalyzing the first step in heme
biosynthesis(19) . As shown in Fig. 3A, heme
deficiency results in significantly reduced amounts of YHG mRNA (about
8-fold by densitometry). Levels return to those of wild type with the
addition of 100 µg/ml
-aminolevulinic acid, which circumvents
the hem1 lesion in the pathway.
Figure 3:
Effect of heme and heme-activated
transcription factors on YHG mRNA levels. RNA was isolated from midlog
phase cells grown in YPGal. The RNA blots were hybridized with the YHG
PCR probe. All cultures were supplemented with 10 µg/ml ergosterol
and 0.1% Tween 80. A, lanes show wild-type strain RZ53-6 (wt), RZ53-6hem1 (hem1), and
RZ53-6hem1 supplemented with 100 µg/ml
-aminolevulinic acid (hem1 + ALA). Setting wild type
as 1.0 for band intensity, the following densitometry results were
obtained: 1.0, 0.12, and 1.41. B, total RNA was isolated from
strains BWG7-1a (wt), LPY22 (hap1),
JO1-1a (hap2), and JS109 (hap1/hap2) grown
under aerobic and anaerobic (N
) conditions. Both parts of
the experiment are from the same blot, and the N
lanes have
been placed underneath the aerobic lanes for comparison. Both aerobic
and anaerobic conditions produce the identical 1.2-kb YHG mRNA. The
ACT1 probe was used as an internal standard for mRNA loading.
Densitometry results for the YHG band (with wild type in air set at
1.0) are: 1.0, 0.46, 0.29, and 0.02 in air and 0.25, 0.10, 0.10, and
0.11 in nitrogen.
Strains deficient in HAP1 or HAP2 also display a decrease in YHG message (2.2- and 3.4-fold by densitometry) compared with that of wild type (Fig. 3B, top). In a strain where both HAP1 and HAP2 have been mutated, YHG mRNA levels drop about 50-fold from wild type, suggesting that the heme/HAP pathway is predominant in the aerobic transcription of YHG. A strain deficient in the heme-induced repressor ROX1 (RZ53-6 rox1) had no change in aerobic YHG gene expression (not shown). The sharp decrease of transcription in the hap1/hap2 strain was surprising, given that there is some expression of YHG even under anaerobic conditions (Fig. 2). To determine if the anaerobic expression of YHG is HAP-independent, we isolated RNA from wild-type cells and from the HAP mutants grown anaerobically. The results, shown in the bottom of Fig. 3B, reveal persistent anaerobic production of YHG mRNA even in the hap1/hap2 double mutant.
To confirm that oxygen/heme
control of YHG mRNA is at the level of transcription, 1.6 kb of the YHG
promoter was fused to the lacZ reporter gene and integrated
into the genome of wild-type, hem1, hap1, hap
2, and hap1/hap2 strains, and lacZ assays were
performed (Table 2). A 4-fold decrease in lacZ activity
was observed in heme-deficient cells compared with wild-type levels.
Strains carrying a mutation in either HAP1 or HAP2 also showed about a
4-fold decrease from wild-type lacZ levels, whereas the hap1/hap2 double mutant produced about one-tenth of the
-galactosidase produced by the wild-type strain. The YHG
promoter/lacZ fusion construct integrated into the wild-type
strain YM4134 also displayed a 3-fold decrease in YHG promoter activity
as the cells exited log phase (Fig. 4) or were subjected to
decreased oxygen tensions (not shown).
Figure 4: Effect of culture density on expression of the YHG promoter/lacZ construct. YM4134 carrying the integrated pYH10 construct was grown in YPGal media and assayed for lacZ activities at the optical densities shown. The lacZ activity points represent the averages of three separate cultures done in duplicate. Individual specific activities never varied by more than 15%.
Figure 5: Effect of carbon source on YHG expression. YM4134 cells were grown in YP medium supplemented with 2% glucose, 2% galactose, 2% raffinose, 4% glycerol, 4% lactate, or 4% ethanol. The latter three cultures were also supplemented with 0.1% galactose for enhanced early growth. Total RNA was isolated from midlog phase cultures, and the subsequent blot was hybridized with YHG. ACT1 probe was used as an mRNA loading standard.
Figure 6:
Northern and Western analyses of YHG
knockout. A, total RNA was isolated and blotted from YM4134 (wt) and the hemoglobin knockout strain YD7 (-yhg). The membrane was hybridized to YHG probe and,
after stripping, with ACT1 probe. B, total protein from 1.5
10
log-phase cells of YM4134 and YD7 was run on
SDS-polyacrylamide gel electrophoresis and probed with rabbit
-YHG
antiserum raised against recombinant YHG expressed in E. coli.
Numbers corresponding to the 52- and 34-kDa molecular mass markers are
shown. The 47-kDa band seen in the wt lane is the predicted
size of the YHG protein(8) .
No phenotypic differences were found between the wild-type and the yhg strains in a variety of conditions tested, including growth on fermentable or non-fermentable carbon sources from 100% oxygen to strict anaerobiosis. When induced to lose its mitochondrial DNA by ethidium bromide treatment(46) , the knockout strain formed petite colonies with no effect on fermentative growth (not shown).
Regulation of the S. cerevisiae flavohemoglobin gene is clearly controlled by cell density and oxygen tension but in a manner different from that of the bacterial globins previously studied. During our efforts to correlate YHG transcriptional activation with oxygen concentration, we were able to determine at what oxygen tension the apparent switchover from aerobic to anaerobic gene expression in S. cerevisiae occurs. The messages for both the aerobic TIF51A and the anaerobic TIF51B are found at 0.1% oxygen. Above this concentration, only TIF51A is present; below this concentration, only TIF51B can be detected. A similar result was seen for the COX5a/b gene pair. This is also the oxygen tension where aerobic activation of the YHG promoter ends and the anaerobic expression of the YHG message begins. Thus, 0.1% likely straddles the concentration of oxygen necessary for heme biosynthesis.
Aerobic expression of YHG is predominantly activated by the HAP1 and HAP2/3/4 transcription factor complexes. An anaerobic system to produce YHG message independent of the HAPs is also present. Although there is a decrease in YHG gene expression as the cells approach anaerobiosis, the effect is not as dramatic as the depletion in YHG mRNA as the cells reach higher optical densities ( Fig. 1compared with Fig. 2). The additional repression of endogenous YHG gene expression upon exit from exponential phase may be due to a combination of factors such as hypoxic conditions, induction of a repressor, or the altered chromatin structure at higher culture densities that has been implicated in repressing most exponential phase mRNA species(47) .
Our results indicate that the YHG promoter/lacZ fusion construct is also regulated by heme and the HAPs, but interesting quantitative differences between Northern and transcriptional fusion analyses exist. The hem1 and hap1/hap2 strains display 4- and 10-fold decreases in lacZ activity relative to wild type, whereas quantitation of Northern analyses indicate a YHG mRNA decrease of 8- and 50-fold for the hem1 and hap1/hap2 strains, respectively ( Fig. 3and Table 2). The drop in lacZ activity as the cells exit log phase is also less dramatic than that of the native YHG message ( Fig. 4compared with Fig. 1). These results raise the possibility that sequences outside of the 1.6-kb upstream region governing the lacZ fusion influence transcription or stability of the endogenous YHG gene product.
The physiological function of hemoglobins in species
other than vertebrate animals has not been well established. The
present data suggest that, despite the high degree of homology between
globins of prokaryotic and eukaryotic microorganisms, each species may
have developed a discrete role for its hemoglobin. Through activation
by bacterial FNR or related transcription factors, hemoglobins of the
strictly respiring Vitreoscilla and A. eutrophus species are probably needed for metabolism when oxygen supplies
are severely limiting or absent(9, 15) .
Overexpression studies in E. coli indicate that the Vitreoscilla globin can increase oxygen uptake and can even
act as a terminal oxidase(4, 5) . An A. eutrophus strain carrying a deletion of the flavohemoglobin gene could not
transiently accumulate nitrous oxide during anaerobic growth using
nitrate as the terminal electron acceptor, suggesting a role of this
protein in the metabolism of a gas other than dioxygen(9) . A
specialized role for the A. eutrophus globin is suggested by
the positioning of the native flavohemoglobin gene on an episomal
plasmid linked to denitrification processes (48) and by the
inability of species that contain homologous globins, such as S.
cerevisiae, to use nitrite as an anaerobic terminal electron
acceptor(49) . Another area of variability between members of
the flavohemoglobin family is in their affinities for oxygen. The
dissociation constant for the C. norvegensis flavohemoglobin
in solution is 2 10
M, making it
one of the most oxygen-avid hemoglobins known(50) . The
dissociation constants for the Vitreoscilla and E. coli hemoglobins are 7.2
10
and 2
10
M, respectively, a difference of at
least 2 orders of magnitude from the C. norvegensis globin,
despite nearly 50% sequence identity in the heme-binding domains of
these proteins(51, 52) .
Clues to the possible function of the S. cerevisiae flavohemoglobin have been uncovered by the gene regulation results presented here. Unlike the Vitreoscilla globin, YHG is probably not involved in facilitating oxygen storage or diffusion during hypoxic electron transport. Contrary to many other genes involved in respiration, the YHG message represents the first known example of a HAP2/3/4-regulated gene that is not glucose-repressed, indicating that the gene may be required in both fermentable and non-fermentable carbon sources. There is an increase in YHG mRNA levels in cells grown under high oxygen tension. This phenomenon can be attributed to increased heme levels, which also stimulate superoxide dismutase and catalase transcription in S. cerevisiae(53, 54) . Flavohemoglobin in S. cerevisiae may also be involved in the detoxification of oxygen. It should be noted, however, that no detectable growth difference is seen between wild-type or yhg strains when grown in 100% oxygen or in the presence of hydrogen peroxide (not shown).
The capacity of the YHG promoter to remain active even during anaerobic growth suggests that the hemoglobin may be necessary in certain anaerobic conditions. A phenotype for the A. eutrophus strain lacking hemoglobin was found only when cells were grown anaerobically(9) . It can be postulated that low levels of YHG might be needed anaerobically to quickly initiate a signal cascade once oxygen becomes available. Activity of the C-terminal reductase domain in YHG could be altered with the binding of heme or heme and oxygen in the N-terminal portion of the molecule. A heme-containing oxygen sensor has been found in the Rhizobium bacteria, where an oxygen-regulated kinase in the N terminus is linked to a heme-binding domain in the C terminus(55) . Biochemical studies of the E. coli flavohemoglobin indicate that flavin reduction in the protein is increased upon deoxygenation, suggesting that the reductase activity may be altered by the availability of oxygen(52) . Further phenotypic studies of the YHG knockout strain and biochemical analyses of the YHG protein should provide insight into the role of flavohemoglobins from facultative eukaryotic microorganisms.