©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Effect of Amino Acid Substitutions in the Conserved Aromatic Region of Subunit II of Cytochrome c Oxidase in Saccharomyces cerevisiae(*)

(Received for publication, November 16, 1995; and in revised form, January 16, 1996)

Michael H. Overholtzer Peter S. Yakowec Vicki Cameron (§)

From the Biology Department, Ithaca College, Ithaca, New York 14850

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mitochondrial encoded subunit II of cytochrome c oxidase carries the metal center, which acts as the initial acceptor of electrons from cytochrome c. Among the conserved features of this protein is a region in which five aromatic and three non-aromatic amino acids are conserved in a wide variety of organisms. This aromatic region has been postulated to be involved in transfer of electrons from the copper center in subunit II to the remaining metal centers of cytochrome oxidase in subunit I. To test the functional importance of two conserved, aromatic tryptophan residues and one conserved, non-aromatic glycine residue, yeast strains with alterations at these positions were characterized. The strains with altered codons were tested for their ability to carry out cellular respiration, for their growth rates on non-fermentable carbon sources, and for their cytochrome c oxidase activity. The results demonstrate that the aromatic character of the tryptophan residues appears necessary for subunit II function, while the conserved glycine can be replaced with other, small, uncharged residues.


INTRODUCTION

Cytochrome c oxidase is the terminal protein complex of the electron transport chain, responsible for transferring electrons from cytochrome c to molecular oxygen. In eukaryotes, the three largest subunits, subunits I, II, and III, are encoded on mtDNA, while the remaining subunits are encoded on nuclear DNA(1, 2) . Aerobic bacteria have only three subunits in a completely functional cytochrome oxidase complex. These prokaryotic subunits are homologous to eukaryotic subunits I, II, and III(3) . It therefore seems likely that subunits I, II, and III of the eukaryotic complex comprise the catalytic core of the enzyme, while the remaining nuclear encoded subunits may be involved in regulation or the assembly of the complex.

Deletion of the subunit III gene from the bacterium Paracoccus denitrificans by in vitro mutagenesis causes disruption in the assembly of the oxidase complex, but removal after assembly does not appear to abolish electron transport and proton pumping activity (4) . These results suggest that subunits I and II are directly responsible for the catalytic activity of the enzyme. In addition, the five metal centers associated with the cytochrome c oxidase complex are bound exclusively by subunits I and II. Heme a, heme a(3), and Cu(B) are associated with subunit I, and subunit II binds a binuclear metal center, Cu-Cu, which is the site of the initial redox reaction with cytochrome c(5, 6, 7) . A better understanding of the mechanism of cytochrome c oxidase function may therefore be achieved by focusing on subunits I and II.

Subunit II is of interest because it is the primary acceptor of electrons from cytochrome c(8, 9) . Subunit II contains several regions of conserved amino acids from organisms as diverse as prokaryotes, fungi, plants, and humans, including two conserved stretches of hydrophobic amino acids, presumed to span the inner mitochondrial membrane, a number of amino acids that act to bind the binuclear copper center, and a highly conserved aromatic region(10) . The aromatic region is a sequence of 10 consecutive amino acids, including five aromatic and three non-aromatic which may mediate electron transfer from the binuclear copper center in subunit II to heme a in subunit I(10) . To determine if a number of these conserved residues are essential for protein function, and in particular, if the aromatic character of two of the conserved amino acids is important, point mutations in the subunit II gene of Saccharomyces cerevisiae that alter two highly conserved aromatic residues and one conserved non-aromatic residue were analyzed. The consequences of these amino acid substitutions with respect to respiratory competence, growth rates, and cytochrome c oxidase activity are reported.


MATERIALS AND METHODS

Strains and Media

The strains of S. cerevisiae used in this study are listed in Table 1. The media used have been described previously(11) .



Isolation of Mutants and Revertants

Isolation of respiration-deficient mutant strains and localization of the mutational event to either nuclear or mitochondrial DNA were performed as described previously(11) . VC20 and VC21 (mata, his4, pet9 (cox2)) were crossed to JM8 (mat alpha, ade1 (^0)) and m5301 (mat alpha, ade1, pet9 (cox2)) was crossed to JM6 (mata, his4 (^0)) to create diploids of the genotype (mata/alpha, +/ade1, +/his4, +/pet9 (cox2)). Multiple isolated diploid colonies, derived from single, respiration-deficient cells, were grown overnight in 5 ml of YPD medium. The contents of each tube were centrifuged, the cells washed with water, and the entire contents of the tube plated on two YEPG plates. One plate was incubated at 18 °C and the other at 28 °C. The plates were examined at intervals, and revertant colonies that arose were colony-purified.

DNA Isolation and Sequencing

For the mutant strains, the entire COX2 gene was amplified from crude yeast cell lysates and sequenced, using a dsDNA cycle sequencing kit from Life Technologies, Inc., as described previously(12) . Although we did not determine the entire sequence on both strands, in each case our sequences precisely matched the reported wild type sequence or contained only a single nucleotide change. The sequencing reactions were repeated on independent, isolated mutant colonies where nucleotides other than wild type were detected. For revertants, the sequence in the region of the original mutation was determined. If an alteration was found at the same site as the original mutation, the second alteration was assumed to compensate for the first. If the mutant codon remained, the entire COX2 gene was sequenced to determine if an alteration elsewhere in the COX2 sequence was responsible for restoration of function.

Genetic Analysis of Revertants

Diploid revertants were sporulated, the asci dissected, and the phenotypes of the resulting spores were determined. If the ability to respire segregated 2:2, as would be expected for a mitochondrial suppressor or for a nuclear suppressor linked in cis to the wild type PET9 locus, a respiration-proficient spore of the appropriate mating type was crossed to a ^0 strain. The resulting diploids were again sporulated, the asci dissected, and the phenotypes of the spores determined. Sporulation of these diploids, which no longer carry the mutant pet9 allele, allowed direct localization of the reversion event to mitochondrial DNA (all four spores grow on YEPG) or to nuclear DNA (2:2 segregation of the ability to grow on YEPG).

Biochemical Analysis

Mitochondria were isolated as described previously(13) . Cytochrome oxidase activity assays were performed on mitochondria isolated from cells grown in YPGal as described(14) .


RESULTS

Isolation and Characterization of Strains with Mutations in Subunit II

Two strains with mutations in subunit II, VC20 and VC21, were isolated as described previously(11) . In addition, a third strain, m5301, bearing a mutation in cox2 was obtained from a collection isolated by Weiss-Brummer et al.(15) . The VC20 and VC21 mutations were assigned to the COX2 locus since crosses of these respiration-deficient strains to the strain DS302 conferred the ability to respire to the diploid. DS302 has lost most of its mitochondrial DNA but retains 3.5 kilobase pairs of wild type mitochondrial DNA including the entire wild type COX2 locus (16) . The mutation in m5301 was mapped in a similar fashion(15) .

The entire cox2 gene was amplified and sequenced in each of these mutant strains. In m5301, codon 121 has been altered from GGA (glycine) to GAA (glutamic acid), while in VC20, codon 126 has been changed from TGA (tryptophan) to TGT (cysteine). VC21 carries two mutations, one that converts a non-conserved TAT (tyrosine) codon at position 59 to AAT (asparagine) and one that converts codon 124 TGA (tryptophan) to AGA (arginine). The mutation at codon 59 is likely to be irrelevant since this position is not conserved and since our reversion analysis (see below) demonstrates that recovery of respiratory function depends only on the TGA 124 codon. As shown in Fig. 1, the three mutations of interest are located in the conserved aromatic region of the gene. To determine if each of these residues is absolutely essential for activity and if the aromatic character of the two tryptophan residues is important for their function, derivatives of each of the mutants which had spontaneously recovered respiratory function were isolated and the alterations in these revertant strains were analyzed.


Figure 1: Schematic diagram of the COXII protein. The aromatic region of the protein has been expanded to show the position of the mutations in the strains analyzed. Conserved amino acids are marked with stars.



Isolation of Respiration-proficient Revertants

The three cox2 mutant strains are respiration-deficient due to cox2 mutations, but they also contain a mutation in the nuclear gene pet9 that affects respiratory function. PET9 encodes an ADP/ATP translocator and mutations in this gene are lethal in combination with and ^0 mutations(17) . By isolating mutations in a pet9 background, we ensure that all derivatives that lack cytochrome oxidase activity will be due to point mutations within genes affecting oxidase activity rather than to loss of most or all of the mt DNA.

Since the mutant pet9 allele also causes respiration deficiency, the presence of this mutation requires that respiration-proficient revertants be isolated from a diploid heterozygous for the pet9 mutation. Consequently, each of the mutant strains was crossed to a ^0 strain to give a diploid of the following genotype (mata/alpha, +/ade1, +/his4, +/pet9 (cox2)). Recovery of respiration in such a diploid requires reversion only of the cox2 mutation. For each mutant strain, isolated diploid colonies were grown overnight in glucose-containing medium, and the cells spread on two agar plates containing glycerol and ethanol. The three respiration-deficient diploids are incapable of growing on this medium, and therefore any colonies that arise from these diploid cells must carry a dominant suppressor of the original mutation. The pairs of plates were incubated at 28 °C and 18 °C and examined periodically over a 4-week period. Colonies that arose were colony-purified and subjected to further analysis. To ensure that the analysis did not include cells derived from a single mutational event, only one colony arising on a plate on a particular day was chosen for further analysis, unless the two colonies differed significantly in size. Since colony size is a reflection of growth rate, colonies of different size are unlikely to be derived from sibling cells. Furthermore, colonies that arose on different days are also unlikely to be siblings. This conjecture is based on evidence that early arising and late arising mutations, seen under selective conditions in both Escherichia coli and S. cerevisiae, are not due to the same mutational mechanisms and exhibit a spectrum of nucleotide changes(18, 19) . Therefore, colonies arising on different days that were derived from the same original culture were retained for further analysis.

The frequency with which revertants appeared was variable in the three mutant strains. From 60 5-ml cultures of m5301D, 26 revertants were identified (1.4 times 10 ). Twelve revertants were identified from 40 5-ml cultures of VC21D (1 times 10), while a very large number of revertants (more than 100) appeared on plates derived from 40 5-ml cultures of VC20D (1 times 10). 51 of the latter were ultimately characterized by sequencing.

Sequence Analysis of the COX2 Gene in the Revertant Strains

The entire COX2 gene from each revertant strain was amplified using the polymerase chain reaction and oligonucleotide primers flanking the gene (-33 to -10 and 1013-1036). Portions of the polymerase chain reaction samples were precipitated and sequenced directly in the region of interest. For mutants m5301 and VC21, all reversion events occurred in the original mutant codon. As shown in Table 2, for m5301 (G121E), 17 of the revertants recovered the wild type glycine codon, eight carry an alanine codon and one carries a serine codon. The revertant bearing the serine codon actually has three altered nucleotides. The nucleotide sequence which is ATT GGA (Ile-Gly) in the wild type and ATT GAA (Ile-Glu) in the mutant is changed to ATA TCA (Ile-Ser) in the revertant strain. For VC21 the wild type tryptophan codon is restored in all 12 revertants. We also sequenced three independent revertants of VC21 in the region of codon 59, where the mutant bears a AAT codon (asparagine) rather than the wild type TAT (tyrosine). In all three of these revertants, the mutant AAT codon remains, indicating that this alteration is compatible with subunit II function. For VC20, 51 revertants were characterized, 30 of which were derived from independent cell cultures. The remaining 21, isolated from cultures from which a revertant had already been identified, either arose later in the selection process or gave colonies of different size. As shown in Table 2, 37 of the 51 revertants characterized carry an alteration at the original mutant codon. Of the 37, 23 now carry a tyrosine codon, 5 encode phenylalanine, and 9 encode the wild type amino acid, tryptophan. All of these amino acids are aromatic in nature. Interestingly, four of the codons are TGG, the only tryptophan codon used in the standard genetic code, but which is not present in the coding sequence for any protein encoded on mtDNA(21, 22) . This codon has only been identified in three positions in mitochondrial genome in yeast, within introns of subunit I of cytochrome oxidase and cytochrome b(22) .



Fourteen of the revertants retain the mutant cysteine codon. Since this codon leads to respiration deficiency in the original mutant strain, the sequence of the remainder of the COX2 gene was determined for two of the cysteine revertants. For both of these, the remainder of the gene is wild type, indicating that the alteration responsible for recovery of function resides outside the COX2 locus.

Genetic Analysis of Revertants with Second Site Suppressor

In two of the VC20 (W126C) revertants that retain the mutant cysteine codon, the cysteine codon was shown by DNA sequencing to reside within an otherwise wild type COX2 gene. To determine if recovery of function in these strains and some of the other cysteine codon carrying strains was due to an alteration of nuclear or mtDNA, respiration-proficient diploid cells from five of these revertants were sporulated and the resulting asci dissected onto glucose containing agar plates. The phenotypes of the resulting spores were then determined. The nuclear markers segregated 2:2 as expected, but almost all the spores derived from the respiration-proficient diploids were now respiration-deficient. Such a result is inconsistent with either a nuclear or mitochondrial suppressor, but suggests that the respiration-proficient phenotype of the diploid is unstable and can only be maintained on selective medium.

To test this hypothesis, the original wild type parent, JM22, was crossed to a ^0 strain to create a wild type diploid parent, isogenic to the diploid revertants. The diploid parent, JM22D, and a number of VC20 diploid revertant strains were plated under non-selective conditions, on YPD plates, to obtain single colonies. These colonies were then replica-plated to YEPG plates and grown at 28 °C to assess the stability of the respiration-proficient phenotype. As shown in Fig. 2, for the wild type parent and two other VC20 revertants, one carrying a phenylalanine codon and one carrying a tyrosine codon, essentially all the colonies grow on YEPG. In contrast, for two independent cysteine codon carrying mutants, essentially all the colonies fail to grow on YEPG. Clearly the respiration-proficient phenotype of these revertants can be maintained only under selective pressure.


Figure 2: Stability of the respiration-proficient phenotype in isogenic wild type and revertant strains of VC20 (W126C). Cells were grown on non-selective medium (YPD), then replica-plated to selective medium (YEPG) and the plates incubated for 2 days.



The most likely explanation for all spores being respiration-deficient is that the diploid revertant strain is heteroplasmic, containing two different mitochondrial genomes. One of these genomes would be the original mitochondrial DNA, bearing the VC20 mutation, while the other would be a genome from which most of the mitochondrial DNA has been lost, but on which the compensatory change resides. While both are maintained under selective pressure, the heteroplasmic condition is unstable; therefore, when the cells are grown in glucose, i.e. under non-selective conditions, one or the other of the mitochondrial genomes is rapidly lost, resulting in cells that are respiration-deficient(23) . Since the two mitochondrial genomes segregate from one another at high frequency, it should be possible to identify spores that carry only the suppressor genome and not the genome bearing the VC20 mutation. Identification of such a spore is under way to characterize the nature of the suppressor mutation.

Sibling Status of Revertants

Since several of the revertants were isolated from the same culture, we determined if they carried identical nucleotide changes in the COX2 gene. As shown in Table 3, for m5301 and VC20, in many cases the strains carried different codons at the position of interest. For m5301, three pairs of potential sibling revertants were identified. In two of the three pairs, the members of the pair carry different codons indicating that they are not siblings. When VC20 revertants arose from the same culture but were identified on different days, at different selection temperatures, or which gave colonies of different sizes, the revertants were often not identical. These results support the hypothesis that such revertants are not siblings derived from a single mutagenic event.



Phenotypes of Revertant Strains

To determine if the amino acid substitutions in the revertant strains had physiological effects, diploid representatives of each class were streaked on YEPG plates and their growth rates at 18, 28, and 37 °C were compared. As shown in Fig. 3, for the amino acid substitutions at position 121, while the diploid mutant m5301D is unable to grow, the diploid revertants derived from it grow like wild type at all three temperatures.


Figure 3: Effect of substitutions at codon 121. Isogenic diploid strains were grown on non-fermentable carbon sources at 18, 28, and 37 °C.



When a similar growth experiment was carried out for revertants bearing substitutions at codon 126, diploid revertants bearing either a wild type tryptophan codon or a phenylalanine codon grow comparably to wild type, while cysteine and tyrosine codon carrying revertants grow more slowly. As shown in Fig. 4, two independent cysteine codon revertants grow at different rates, suggesting that one of these strains might have incurred a second mutation which also affects the ability to grow on non-fermentable carbon sources, or that the nature of the suppressor in these two strains is different.


Figure 4: Effect of substitutions at codon 126. Isogenic diploid strains were grown on non-fermentable carbon sources at 18, 28, and 37 °C.



Since growth on agar plates is a crude measure of respiratory ability, mitochondria were isolated from diploid strains bearing amino acid substitutions and cytochrome c oxidase activity was measured. As shown in Table 4, replacement of glycine at codon 121 by alanine or serine appears to have little effect on cytochrome oxidase activity. At codon 126, strains bearing TGA and TGG tryptophan codons and TTT phenylalanine codons have wild type levels of cytochrome oxidase activity. On the other hand, replacement with a TAT tyrosine codon leads to an 80% reduction in activity. Two different cysteine codon carrying strains were analyzed. One of these, MODR30D has about 18.5% of the cytochrome oxidase activity seen in wild type while the other, TTDR4, has only 2.8% of wild type activity. These results are consistent with the growth rates determined on YEPG plates, where TTDR4 (cysteine) grew most slowly and MODR30D (cysteine) and MODR29B (tyrosine) grew at intermediate rates.




DISCUSSION

The three mutant strains characterized bear alterations in conserved amino acids found in the aromatic region of the subunit II protein, which has been suggested to play a role in electron transfer (10) . However, reversion analysis suggests that some amino acid substitutions at these three sites are permissible.

VC21

All 12 of the revertants isolated from VC21 carry a wild type tryptophan codon. Furthermore, in three independent revertants, a secondary mutation at codon 59 in VC21, is still present. These results indicate that the alteration at codon 59 does not impair subunit II function. However, the significance of all revertants restoring the wild type tryptophan codon at position 124 is less clear. In the mutant, the wild type TGA codon has been changed to AGA, which encodes arginine. This is an extreme substitution, replacing an aromatic amino acid with one bearing a positive charge. Serine, arginine, lysine, threonine, glycine, and tryptophan codons can be derived from the mutant codon by single nucleotide changes. Of these, only tryptophan codons have been detected. This result suggests that replacement of tryptophan by positively charged or neutral amino acids is incompatible with function. However, it remains unclear if tryptophan is the only acceptable amino acid at this position or if the only requirement is for an aromatic amino acid, since no codons for other aromatic amino acids can be derived by a single nucleotide change.

VC20

In VC20 the conserved tryptophan 126 codon is converted to cysteine. If an aromatic amino acid is essential at this position, it is reasonable that replacement of tryptophan with cysteine renders subunit II non-functional. The reversion analysis supports the hypothesis that an aromatic amino acid is required at this position. The codons detected at this position in the revertant strains are those for tryptophan, phenylalanine, and tyrosine. Among the same site revertants, representatives bearing TGA and TGG tryptophan codons and TTT phenylalanine codons have levels of cytochrome oxidase activity that are indistinguishable from wild type. The only TGG codons found within yeast mitochondrial DNA are located in the intron-encoded maturases(21, 22) . Presumably, a standard wobble association with the tRNA, which recognizes TGA, allows efficient translation of the TGG codons. The single aromatic ring on phenylalanine appears to substitute completely for the two rings found in tryptophan. In contrast, replacement of tryptophan by tyrosine, which differs from phenylalanine by the addition of a hydroxyl group, reduces cytochrome oxidase activity by more than 80%. Apparently, introduction of the hydroxyl group in tyrosine is sufficient to reduce electron transfer through the protein. Single nucleotide alterations in the mutant codon could yield serine, arginine, phenylalanine, tyrosine, and tryptophan codons (excluding cysteine codons, which clearly require a second-site compensatory change). All three aromatic codons were observed, but none for serine or arginine. Taken together with the fact that introduction of a hydroxyl group to phenylalanine reduces activity significantly, the aromatic character of position 126 appears to be critical for subunit II function.

Fourteen revertants were isolated that still carry the mutant cysteine codon. The complete sequence of subunit II has been determined for two independent cysteine revertants. In both cases the remainder of the gene is wild type. Therefore, a compensatory change must have arisen outside the COX2 locus. For five of the cysteine codon revertants, the respiration-proficient phenotype is highly unstable and yields almost all respiration-deficient spores. These results are consistent with the diploid revertants being heteroplasmic, bearing both the genome carrying the VC20 mutation and another, genome, which bears a suppressor of this mutation. Either genome alone leads to respiration deficiency, since one bears the cox2 mutation, while the other carries the suppressor but is missing other mitochondrial genes that are required for respiration. It is not yet known if all the cysteine codon revertants are suppressed by a similar mechanism. At least one of the revertants, TTDR4, has a very slow growth rate relative to other cysteine codon revertants, but slow growth may be due to the presence of another unrelated mutation that also affects respiratory function. One possible candidate for the suppressor mutation is one of the tRNA genes. Two mitochondrial genes for tRNA, bearing the same anticodon have been reported(24) , so mutation of one of these to recognize the cysteine codon but to insert an aromatic amino acid is a potential mechanism of suppression. Characterization of these suppressors is under way.

m5301

The mutation in m5301 converts a conserved glycine codon to one encoding glutamic acid. This is a radical substitution, inserting a large, negatively charged residue for a small neutral one. Although the glycine is conserved, it is not aromatic, suggesting that if the complex utilizes aromatic rings to shuttle electrons through this region, the conserved glycine may be important for maintaining the structure of the region rather than electron transfer per se. If this hypothesis is correct, amino acid substitutions at this position that do not dramatically alter the structure of the region may be compatible with function. Reversion analysis supports this hypothesis since codons for both serine and alanine, which encode small, uncharged amino acids, are detected in revertant strains. Furthermore, levels of cytochrome oxidase activity from strains bearing these codons appear to be essentially wild type. Other amino acids that could be derived by single nucleotide changes in the mutant codon include glutamine, lysine, and aspartic acid, but none of these are seen. Codons for these amino acids represent four of the seven possible which can be derived by single nucleotide changes, besides those encoding the non-functional glutamic acid codon and stop. Our results suggest that replacement of glycine with other small neutral amino acids has little affect on the activity of the protein, while replacement with larger or charged amino acids is incompatible with function.

``Sibling'' Revertants

A number of revertant strains, derived from the same culture but identified at different temperatures, after a different number of days of growth, or from colonies of different sizes, were sequenced and compared. A number of these were not siblings since different codons were identified at the position of interest. For m5301, two of three pairs of potential siblings were different than one another. For VC20, seven cultures gave rise to multiple revertants. For only one of these were all the revertants identical. For those bearing the same codon, it is impossible to determine if their changes arose independently of one another. However, the data strongly indicate that revertants arising from the same culture can be different from one another and can contribute important information about the frequency with which specific revertant types arise.

In summary, the analysis has demonstrated that conserved amino acids in the aromatic region of subunit II are important for its function. Reversion analysis suggests that the conserved glycine at position 121 may be important for structural reasons, since the only substitutions detected were with amino acids similar in size and nature. In addition, the aromatic character of the tryptophans at positions 124 and 126 is important for the function of the protein, and they cannot be replaced by non-aromatic amino acids. Tryptophan 126 can be replaced, without loss of function by phenylalanine, and replacement by tyrosine results in reduction but not elimination of cytochrome c oxidase activity. These results are consistent with the hypothesis that the aromatic region functions in electron transfer.


FOOTNOTES

*
This work was supported by Research Grant MCB-9317071 from the National Science Foundation (to V. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 607-274-3575; Fax: 607-274-3474; cameron{at}ithaca.edu.


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