Saccharomyces cerevisiae Cytochrome c Oxidase Subunit V by

Vol. 8, No. 10

MOLECULAR AND CELLULAR BIOLOGY, OCt. 1988, p. 45374540 0270-7306/88/104537-04$02.00/0 Copyright © 1988, American Society for Microbiology

Differential Regulation of the Two Genes Encoding Saccharomyces cerevisiae Cytochrome c Oxidase Subunit V by Heme and the HAP2 and REOJ Genes C. E. TRUEBLOOD, R. M. WRIGHT, AND R. 0. POYTON* Molecular, Cellular, and Developmental Biology, Campus Box 347, University of Colorado, of Department Boulder, Colorado 80309 Received 28 March 1988/Accepted 17 June 1988

In Saccharomyces cerevisiae, the COXSa and COXSb genes encode two forms of cytochrome c oxidase subunit Va and Vb. We report here that heme increases COX5a expression and decreases COX5b expression and that the HAP2 and REOI genes are involved in positive regulation of COX5a and negative regulation of COX5b, respectively. Heme regulation of COX5a and COX5b may dictate which subunit V isoform is available for assembly into cytochrome c oxidase under conditions of high- and low-oxygen tension. V,

buffer (12) containing 20 RI of 0.1% sodium dodecyl sulfate and 20 RI of chloroform and broken by vortexing for 10 s. Under these cell breakage conditions, 3-galactosidase is effectively released from the cell (4). Moreover, even though 13 amino acids of the Va and Vb leader peptides are at the NH2 termini of the V.-7-galactosidase and Vb-,-galactosidase fusion proteins, respectively, it is clear from Pgalactosidase assays performed on mitochondrial and cytosolic cell fractions that little or none of the P-galactosidase synthesized resides in mitochondria (C. Trueblood and D. Schultz, unpublished observations). When the hem] mutant strains (CT155-7A and CT155-7B) were supplemented with b-ALA, COX5a-lacZ expression was increased 10- to 20-fold and COXSb-lacZ expression was decreased about five- to sevenfold (Table 1). These results indicate that heme (or one of the precursors in the heme biosynthetic pathway) acts as a positive effector of COX5a expression and a negative effector of COXSb expression. To identify genes that are required for positive heme regulation of COXSa, we have investigated the effects of hapi and hap2 mutations on expression. The HAP] (CYPI) and HAP2 gene products are involved in heme activation of CYCI (the gene encoding iso-1-cytochrome c) through their action at the upstream activation sites UAS1 and UAS2, respectively, of CYCI (8, 15, 16). The hapl::LEU2 mutation resulted in a small decrease (about 1.5-fold) in COXSa-lacZ expression and had little or no effect on COXSb-lacZ expression in aerobically grown heme-proficient cells. The significance of these small changes is unclear. In contrast, the hap2-1 mutation in strain LGW1 had a dramatic effect on expression of COXSa-lacZ, reducing it about eightfold relative to expression in the isogenic HAP2+ strain, BWG1-7A, but had no effect on the expression of COXSb-lacZ (Table 2). We conclude that the HAP2 gene product acts as a positive regulator of COX5a and that it is not involved in heme regulation of COX5b. A comparison of COX5a 5' sequences with the recently defined regions 1 and 2 of CYCI UAS2 (5) reveals a COXSa sequence ([-147] TGATTGGc [-140]) that has a single transition change compared with the region 1 consensus, TNATTGGT. Further work is necessary to determine whether this sequence is involved in HAP2mediated regulation of COXSa. To identify trans-acting genes involved in negative heme regulation of COXSb, we have investigated the effects of a

The recent discovery of different forms (isologs) for some of the nuclear-encoded subunits of cytochrome c oxidase in mammals and yeasts has led to the hypothesis that isologs play a role in altering the functional properties of the holoenzyme (2, 9, 10, 21). In Saccharomyces cerevisiae, the only cytochrome c oxidase subunit for which isologs have been identified is subunit V (14); the two isologs, Va and Vb, are 66% similar in amino acid sequence and are encoded by the unlinked divergent genes COXSa and COX5b (3). Either Va or Vb can provide a required subunit V function to the enzyme, but the expression of COXSa and COX5b differs dramatically (2, 21). To gain insight into why yeast cells have maintained two subunit V genes, we have initiated studies to compare the regulation of COXSa and COXSb and to examine the functional properties of cytochrome c oxidase containing Va or Vb. In this paper, we demonstrate that COXSa and COXSb are inversely regulated by heme and show that the HAP2 and REOJ genes are involved in positive regulation of COX5a and negative regulation of COX5b, respectively. The effect of heme deprivation on expression of COXSa and COX5b was tested with strains carrying hem] mutations. hemi mutant strains are heme deficient because of an inability to synthesize b-amino levulinic acid (8-ALA) (7, 23); they can be made heme proficient by the addition of b-ALA to the growth medium. RNA blot analysis (Fig. 1) demonstrated that a hemi mutant strain grown in the presence of b-ALA (lanes 1 and 3) had increased steady-state levels of COXSa transcripts and decreased levels of COX5b transcripts compared with transcript levels of the same strain grown in the absence of b-ALA (lanes 2 and 4). Additional evidence for inverse regulation of COXSa and COX5b by heme was provided by ,-galactosidase activities measured in hemi mutant strains transformed with COXSalacZ and COX5b-lacZ fusion genes, carried on the 2,umbased plasmids pCT5aL and pMC5bL, which were constructed as described previously (21). Cultures carrying these plasmids were grown to exponential phase in synthetic glucose medium (2) lacking uracil (to select for the plasmids) but containing 40 ,ug of histidine, 40 ,ug of tryptophan, 100 Kg of leucine, and 20 ,ug of ergosterol per ml and 0.25% Tween 80. After being harvested, cells were suspended in 1 ml of Z *

Corresponding author. 4537

4538

NOTES

MOL. CELL. BIOL.

TABLE 2. Regulation of COX5a by HAP2 Strain' .'

51-

genotype

U

BWG1-7A LGW1

FIG. 1. Heme proficiency increases COX5a transcript abundance and decreases COX5b transcript abundance. Poly(A)+ RNA (20 ,ug) was from the heml mutant strain GT38-7A (23) grown in YPD medium containing 0.5 mM 6-ALA (lanes 1 and 3) or lacking 8-ALA but containing 0.1% Tween 80 and 20 ,ug of ergosterol per ml to allow growth under heme-deficient conditions (lanes 2 and 4). Lanes 1 and 2 were hybridized to a radiolabeled probe specific for COX5a mRNA (a 4-kilobase EcoRI fragment of COX5a nick translated in the presence of [32P]dATP). Exposure time was 24 h. Lanes 3 and 4 were hybridized to a radiolabeled probe that had a 130nucleotide region complementary to COX5a mRNA and two regions (of 90 and 550 nucleotides) complementary to COX5b mRNA (a 3.7-kilobase BamHI-ClIal fragment of COX5ab. which is a chimeric gene constructed from COX5a and COX5b, as described previously [21], was nick translated in the presence of [32P]dATP). Exposure time was 7 days. Poly(A)+ RNA was isolated from aerobically

growing exponential-phase cultures, separated electrophoretically on a 1.2% agarose gel containing formaldehyde, blotted to nitrocellulose, and hybridized to radiolabeled probes by methods described previously (24).

recently described recessive mutation, reol (C. E. Trueblood and R. 0. Poyton, Gene, in press), on expression in heme-proficient and heme-deficient cells. Recessive mutations in the REOI gene permit a strain deleted for COX5a to grow on nonfermentable carbon sources by increasing the intracellular level of the Vb polypeptide. In a HEMJ + reol mutant strain, expression of the COX5b-lacZ fusion gene about eightfold higher than in an isogenic REOJ + strain, whereas expression of the COX5a-lacZ fusion gene was unaffected (Table 3). The involvement of the REOI gene product in heme regulation of COX5b was demonstrated by 3-galactosidase assays performed on hemi reol and hemni REOI + mutant strains (Table 3). The addition of b-ALA to the hemi mutant strains repressed COX5b-lacZ expression only when the REOI gene was intact, indicating that negative regulation of COX5b-lacZ by heme requires the REOI was

on

COX5a and COX5b expression

Strain (genotype)"

Heme phenotype"

CT155-7A (his4 trpl ura3 hemi) CT155-7B (his4 trpl leu2 ira3 heml)

Heme-

Heme+ Heme

Heme+

1-Galactosidase activity' (OD42,JOD),, per min) for: COX5ci-lac Z COX5Sb-lacZ 7.5 70 5 104

15 2.1 8 1.4

"CT155-7A and CT155-7B were derived from a tetrad of CT155, a strain resulting from the fourth backcross of strain GT38-7A (MA Tot he,nl obtained from J. Mattoon [23]) to our laboratory strain JM43 (MA To his4-580 trpl-289 leui2-3,112 ura3-52). bThe heme+ designation indicates the addition of 100 ,ug of 5-ALA per ml. This supplement bypasses the heinl defect and allows synthesis of heme. ' Measured and calculated by the method of Miller (12). Values are averages of triplicate measurements of activity from cultures inoculated with 5 to 10 independent transformants; the standard deviation of triplicate measurements was 20% or less in each case. OD, Optical density.

COX5a-IacZ

COX5b-1acZ

166 19

3.6 3.3

HAP2

hap2-l

' BGW1-7A and LGW1 are isogenic strains obtained from L. Guarente; each strain carries adel-100, his4-519, leu2-3,112, and ura3-52. The hap2-1 allele in strain LGW1 was isolated by screening for mutants of BWG1-7A that expressed only a low level of ,-galactosidase from a CYCI-lacZ fusion gene lacking UAS1 sequences (8). h Measured as described in the text and Table 1, footnote c, except that Tween 80 and ergosterol were not included in the medium but 40 1Lg of adenine per ml was included. The standard deviation for triplicate measurements of ,B-galactosidase was 10% or less in all cases. Similar results were obtained in three additional experiments. OD, optical density.

gene. The positive regulation of COX5a-lacZ by heme was not affected

by the reol mutation. Our observations and the demonstrated requirement for oxygen in heme biosynthesis (11) lead us to propose that oxygen concentration is the environmental signal and heme is the intracellular effector for differential regulation of COX5a and COX5b. According to our model (Fig. 2), when heme is present, the HAP2 gene product activates COX5a transcription and the REOI gene product represses COX5b transcription. As a consequence of this regulation, in aerobically grown heme-proficient cells, production of Va is much greater than that of Vb. In contrast, when intracellular heme is absent or low (in hemi mutant strains or wild-type cells grown anaerobically) (11), HAP2 does not activate COX5a expression, nor does REO1 repress COX5b expression. Consequently, cells should produce as much Vb as Va or more Vb than Va. Our proposals concerning positive TABLE 3. Effects of REOI on negative regulation of COX5b by heme ,B-Galactosidase activity'

Strain'

CT149-3C CT149-3D CT224-16A

Relevant genotype

HEMI REOI HEM] reol hemn] REOI

CT224-16D heini reol TABLE 1. Effects of heme

,B-Galactosidase activity' (OD42(VOD600 per min) for:

Relevant

Heme phenotype'

HemeHeme+ HemeHeme+

(OD42(VOD600 per min) for: COX5b-lacZ

COX5a-IacZ

2.0 15.4 3.4 0.9 5.9 7.5

97 75 3.6 61 2.7 50

"CT149-3C (leu2-3.112 ura3-52 Akcox5a reo-4) and CT149-3D (his4-580 leii2-3,112 ura3 -52) are isogenic with JM43 (2). The reol4 allele was originally isolated in strain CT4 (Trueblood and Poyton, submitted), a spontaneous mutant of GD5a-4 (his4-580 trpI-289 leii2-3,112 ura3-52 Acor5a::URA3) (21) that exhibits increased expression of COX5b. To create the Acox5a allele, the entire coding sequence and flanking sequences on either side of the COX5a gene were deleted. A linear DNA fragment carrying the Acox5a allele was transformed into strain GD5a-4 by the one-step gene disruption method of Rothstein (18). Ura- transformants, which are expected to have the Acox5a: :URA3 allele replaced by Acox5a, were selected with 5-fluoro-orotic acid (1) and confirmed by Southern blot analysis. CT224-16A (his4 leu2 heml ACOX5a) and CT224-16D (his4 leu2 ura3 ACOX5a heml reol) were derived from a tetrad of strain CT224, which was constructed by crossing CT155-7A (Table 1. footnote a) and CT149-4D (leu2-3,112 ura3-52 Acox5a reo14). b Heme+ and heme- denote the presence and absence, respectively, of b-ALA in the culture medium. ' Measured as described in Table 1, footnote c, except that CT149-3C and CT149-3D cultures were not supplemented with Tween and ergosterol. The standard deviation for triplicate measurements was 10% or less in all cases. Similar results were obtained when this experiment was repeated. OD,

Optical density.

NOTES

VOL. 8, 1988 A. HEME PRESENT

HAP2

REOI I

I o

ACTIVATED

REPRESSED

Yb

Ya

E HEME ABSENT REOI

HAP2

NOT REPRESSED

NOT ACTIVATED

Ya

S

Yb

FIG. 2. Model for heme regulation of COX5a and COX5b. (A) In heme-proficient cells that have been grown aerobically (so that heme can be synthesized), the HAP2 gene product activates expression of COX5a, and the REO1 gene product directly or indirectly represses expression of COX5b. The HAP2-mediated activation of COX5a is likely to be transcriptional, since HAP2 has been strongly implicated in transcriptional activation of CYC1 (16) and COXSa transcript abundance is regulated by heme. Because of the observations that heme decreases COXSb transcript abundance and REO1 is required for heme repression of COXSb, we suggest that REO1 acts at the level of transcription. The positive effect of HAP2 on COXSa expression and the negative effect of REOI on COXSb expression in heme-proficient cells result in a high ratio of Va to Vb. (B) In cells unable to synthesize heme because of mutation or anaerobic growth conditions, the HAP2 and REOI gene products are either absent or inactive. The COXSa gene is not activated, so expression is low relative to that observed in heme-proficient cells. The COXSb gene is not repressed, so its level of expression is high in heme-deficient cells compared to that in heme-proficient cells. Thus, heme-deficient or anaerobically grown cells are expected to produce an amount of Vb greater than or equal to the amount of Va produced. The modes of regulation (positive regulation of COX5a by HAP2 and negative regulation of COXSb by REOI) presented in this model are based on evidence that the hap2-1 and reol4 mutations are loss-of-function alleles of HAP2 and REOJ. hap2-1 is considered a loss-of-function allele because strains carrying the hap2-1 mutation exhibit the same phenotype as strains deleted for HAP2 (L. Guarente, personal communication). reol4 is considered a loss-of-function allele because it is suppressed by an amber suppressor tRNA (Trueblood and Poyton, in press).

regulation by HAP2 and negative regulation by REOJ are based on the COX5-lacZ expression phenotypes of the hap2 and reol mutant strains and the evidence that the hap2-1 and reol4 mutations used here are loss-of-function alleles (Fig. 2, legend). The positive regulation of COX5a by HAP2 and heme described here extends the previous observations that hemedeficient cells have a reduced level of translatable subunit Va mRNA (6) and fail to accumulate subunit Va (19). This mode of regulation is consistent with the role of Va as a subunit of cytochrome c oxidase, a protein complex that requires heme to function and uses oxygen as a substrate. It is more difficult to understand why COX5b is negatively regulated by REOI and heme, since COX5b also encodes a form of cytochrome c oxidase subunit V. The inverse heme regulation of COX5a

4539

and COXSb may have evolved to increase Vb production relative to Va production when oxygen concentration is low. Michael Cumsky (personal communication) and co-workers have observed that COXSa transcripts decrease and COX5b transcripts increase in anaerobically grown cells, and we have observed similar results in ,-galactosidase assays (Trueblood and Poyton, in press). Such regulation would be selected for if yeast cells containing Vb have a growth or survival advantage under conditions in which oxygen is limiting. Regulation of the COX5alCOX5b gene family exhibits both similarities and differences with the CYCJICYC7 gene family. The CYCI and CYC7 genes encode iso-l- and iso-2cytochrome c, two polypeptides with 84% identity (13) that represent 95 and 5%, respectively, of the cytochrome c in aerobically grown yeast cells (20). Both COXSa and CYCI exhibit a strong positive response to heme that is mediated, at least in part, by the HAP2 gene product. In contrast, COX5b and CYC7 differ in their response to heme; COX5b exhibits a negative response mediated by the REOJ gene, whereas CYC7 exhibits a positive response mediated by HAP] (17, 22, 25), as well as a negative response to oxygen that may be heme dependent (25). Despite the dissimilarities in regulation of COXSb and CYC7, the overall effect of heme regulation of the two gene families is similar; COXSa and CYCI produce the predominant polypeptide in heme-proficient aerobically grown cells, whereas COXSb and CYC7 expression is significant (relative to COX5a and CYCI expression) only in heme-deficient or anaerobically grown cells. We speculate that the products of COXSb and CYC7, cytochrome c oxidase subunit Vb and iso-2-cytochrome c, respectively, may be better adapted than subunit Va and iso-1-cytochrome c to a low-oxygen environment and perhaps for interaction with one another. Studies addressing these possibilities are in progress. We thank J. Mattoon for hem] mutant strain GT38-7A, L. Guarente for strains BWG1-7A, LGW1, and BWG1-7A hapldis, M. Cumsky for communication of results prior to publication, John Trawick for useful discussions, and Jan Kersell for help in typing the manuscript. This work was supported by Public Health Service research grant GM30228 from the National Institutes of Health. LITERATURE CITED 1. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197:345-346. 2. Cumsky, M. G., C. Ko, C. E. Trueblood, and R. 0. Poyton. 1985. Two nonidentical forms of subunit V are functional in yeast cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 82: 2235-2239. 3. Cumsky, M. G., C. E. Trueblood, C. Ko, and R. 0. Poyton. 1987. Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V. Mol. Cell. Biol. 7: 3511-3519. 4. Douglas, M., B. Gelier, and S. Emr. 1984. Intracellular targeting and import of an F1-ATPase P-subunit-,-galactosidase hybrid protein into yeast mitochondria. Proc. Natl. Acad. Sci. USA 81:

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NOTES

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