Assembly of the mitochondrial membrane system. Characterization of nuclear mutants of Saccharomyces cerevisiae with defects in mitochondrial ATPase and respiratory enzymes.

Mutants of Saccharomyces cereviaiae showing defects in cytochrome oxidase, coenzyme QH2-cytochrome c reductase, and rutamycin-sensitive ATPase are described. The mutations have been established to be nuclear, based on complementation with a cytoplasmic petite tester strain and 2:2 segregation of tetrads. Genetic analysis indicate the coenzyme QH2-cytochrome c reductase and cytochrome oxidase mutants fall into 9 and 10 different complementation groups, respectively. The mutants also form distinct classes based on absorption spectra of the mitochondrial cytochromes. Two of the ATPase mutants lack detectable F1 ATPase, while the third synthesizes F1 but does not integrate it into a membrane complex. The latter mutant is missing one of the mitochondrially synthesized subunits of the rutamycin-sensitive ATPase complex.


Mutants of Saccharomyces
cereuiaiae showing defects in cytochrome oxidase, coenzyme QH,-cytochrome c reductase, and rutamycin-sensitive ATPase are described.
The mutations have been established to be nuclear, based on complementation with a cytoplasmic petite tester strain and 22 segregation of tetrads. Genetic analysis indicate the coenzyme QH,-cytochrome c reductase and cytochronie oxidase mutants fall into 9 and 10 different complementation groups, respectively. The mutants also form distinct classes based on absorption spectra of the mitochondrial cytochromes. Two of the ATPase mutants lack detectable F, ATPase, while the third synthesizes F, but does not integrate it into a membrane complex. The latter mutant is missing one of the mitochondrially synthesized subunits of the rutamycin-sensitive ATPase complex.
In a previous paper we described a selection procedure for the isolation of mutants of Saccharomyces cereuisiae with specific lesions in enzymes of the respiratory chain and of the mitochondrial ATPase complex (1). Approximately 4000 strains showing lack of ability to grow on the fermentable substrate, glycerol, have been examined and of these a number of nuclear mutants have been found with specific defects in the ATPase, coenzyme QH,-cytochrome c reductase, or cytochrome oxidase. In addition other mutants have been found which appear to be defective in coenzyme Q. Some of the biochemical and genetic properties of the mutants are reported here. MATERIALS AND METHODS The prototrophic strain, Saccharomyces cereuisiae D273-IOB was mutagenized with either nitrosoguanidine or ethylmethanesulfonate (1). The screening procedure has been described elsewhere (1). The mutants we report here have been named to indicate the mutagen used. A mutant whose name is preceded by the letter N was isolated from a nitrosoguanidine-treated stock, while the letter E indicates that the mutagen used was ethylmethanesulfonate. Growth of Cells and isolation of Mitochondria-Cells were inoculated from a slant into 70 ml of 2% galactose medium containing 0.3% yeast extract and the basic salts of Ephrussi and Slonimski (21. After overnight growth at 32" on a rotary shaker, the entire culture was used as an inoculum for 1 liter of fresh '2% galactose medium. The culture was aerated at 32" in a %-liter flask by flushing the medium with air through a gas disperser. After aeration for 17 to 24 hours, the cells were harvested, washed with 500 ml of a buffer containing 0.25 M mannitol, 20 mM Tris-acetate (pH 7.51, and 1 mM EDTA, and suspended in 60 ml of the same buffer.
The properties.

NADH-cytochrome c Reductase
Mutants-Thirty-three mutants were found which had no or highly reduced levels of NADH-cytochrome c reductase activity even though they still exhibited from 20 to 100% of the normal levels of cytochrome oxidase and ATPase. These could be divided into two subclasses. Eighteen mutants had no or less than 5% of the wild type level of coenzyme QH,-cytochrome c reductase. In none of these mutants could the NADH-cytochrome c reductase segment of the respiratory chain be restored by the addition of external coenzyme Q. The mitochondrial activities, spectral properties, and the results of some genetic tests of this class of mutants are summarized in Table I. Although the primary defect of the mutants appears to be in the coenzyme QH,-cytochrome c reductase, spectral analysis of the cytochromes in the (Y region of the spectrum indicates that some of the mutants were completely deficient in cytochrome b, while others had either reduced or near to normal levels of the cytochrome. Representative spectra of the three types are shown in Fig. 1. The spontaneous reversion frequency was determined by plating 10" to lo9 cells on glycerol medium and counting revertant colonies after 5 to 7 days of incubation at 30". With the exception of E2-115 most of the mutants either did not revert or did so at a low frequency. The mutants were also tested for spontaneous conversion to cytoplasmic petites. Cells were plated on solid glucose medium and individual colonies were crossed to a p" strain of the opposite mating type.  mutants of S. cereuisiae that have mitochondrial defects (7,8). Tetrad analysis of the mutants indicated a 2:2 segregation of the phenotype, confirming the nuclear nature of the mutations. The coenzyme QH,-cytochrome c reductase mutants fell into at least nine different groups based on genetic complementation.
The NADH-cytochrome c reductase deficient strains also included mutants in which this activity could be restored by the addition of coenzyme QZ to the assay. All the mutants had nearly identical NADH-coenzyme Q reductase. The mutants also showed the presence of variable levels of coenzyme QH,-cytochrome c reductase (Table II). The stimulation of NADH-cytochrome c reductase could be elicited with either coenzyme QZ or the natural analogue of S. cereulsiae, coenzyme Qs. The reconstituted activity was sensitive to antimycin A (Fig. 2). The cytochrome spectra of all the mutants in this group showed the presence of cytochromes c, 6, a, and a,. A few strains were found to be capable of growth on glycerol when coenzyme Q, was added to the growth medium. Although we have not examined the biochemical lesions of this group of mutants further, it appears to consist of strains defective in coenzyme Q.
All the presumptive coenzyme Q mutants showed a 2:2 segregation in tetrad analysis and variable degrees of stability, both with respect to reversion to wild type and conversion to cytoplasmic petites (Table II). At least seven distinct complementation groups were found.
The mitochondrial products elaborated by the NADH-cytochrome c reductase mutants were examined by slab gel electrophoresis in the presence of sodium dodecyl sulfate. The results of these analyses indicated that most of the mutants were capable of forming all the normal products of mitochondrial protein synthesis. Two of the mutants (N5-26 and N6-70), however, had one of the major mitochondrially synthesized proteins missing (Fig. 3). Interestingly, both of these strains were completely deficient in cytochrome b.

Cytochrome Oxidase
Mutants-Among the specific nuclear mutants, approximately 20 were found to have defective cytochrome oxidase. The properties of some of these strains are listed in Table III. The specific activities of cytochrome oxidase in the muta.its chosen were 5% or less of the wild type. Based on their spectral properties, the mutants either had no detectable LY band in the a and a3 region of the spectrum or had highly reduced levels of these cytochromes.
Some representative spectra of the cytochrome oxidase mutants are shown in Fig. 4.
The genetic tests for revertants and for cytoplasmic petite production again indicated a broad range of stabilities. A few mutants (N5-114, N8-105, E2-163, E4-238) degenerated to cytoplasmic petites at a high frequency. Most of the strains, however, were stable (Table III). Tetrad analysis of the mutants indicated a 2:2 segregation of the meiotic spore progeny and complementation tests yielded at least 10 different genetic groups.
It is unlikely that the deficiency in cytochrome oxidase is due  (Table  IV). Those strains in which the incorporation of the radioactive precursor was lower corresponded to strains which produced pat a high frequency. The protein patterns of the mitochondrial products-synthesized by most of the cytochrome oxidase mutants were identical with the wild type. Some of the mutants, however, failed to make one of the proteins; these all lacked spectral cytochromes a and a,.
ATPase Mutants-Three mutants were found to have no rutamycin-sensitive ATPase activity in the mitochondria. These mutants also had considerably reduced levels of NADHcytochrome c reductase and cytochrome oxidase (Table V). In order to test for the presence of F,' ATPase, the mitochondrial and post-ribosomal fractions were assayed for ATPase in the absence and presence of antiserum to F, (Table VI). E2-126 and N9-84 had no detectable F, activity in the post-ribosomal fractions, nor were there any significant antiserum-sensitive units in the mitochondria.
The low hydrolytic activity seen in the mitochondria may be due t.o nonspecific phosphatases or to a non-mitochondrial ATPase contaminant. N9-168 also had no significant rutamycin-or antiserum-sensitive units in the mitochondria.
This mutant, however, had a high specific ATPase activity in the post-ribosomal supernatant that was inhibited by F I antiserum and therefore presumably represents mitochondrial F, ATPase. Conversion to cytoplasmic petites was less than 1% for N9-84 and E2-126 and 8% for N9-168.
N9-84 and E2-126 synthesized all the mitochondrial products seen in wild type. N9-168, however, was completely deficient in one of the proteins. The missing polypeptide (Band 2 in Fig. 3) corresponds to subunit nine of the rutamycin-sensitive ATPase complex which was previously shown to be a product of mitochondrial protein synthesis (9). DISCUSSION Three enzymes of the mitochondrial inner membrane, the rutamycin-sensitive ATPase (lo), cytochrome oxidase (1 l-13), and coenzyme QH,-cytochrome c reductase (14), have been shown to consist of subunit polypeptides, some of which are synthesized in mitochondria and others on cytoplasmic ribosomes. An understanding of how the two protein synthesizing systems interact and how the polypeptide components are integrated into the functional enzyme complexes is one of the current goals in studies of mitochondrial biogenesis. An approach to this problem is through the use of mutants in which defective subunit proteins lead to a blockage of the assembly process. The detection of partially synthesized enzyme intermediates should permit the sequence in which the subunits are integrated to be reconstructed. With this aim in mind, we have sought to isolate strains of S. cereuisiae in which the defects would be restricted to a single mitochondrial enzyme.
The mutants reported in this study appear to be specific for either ATPase, cytochrome oxidase, or coenzyme QH,-cytochrome c reductase. The complementation test with a cytoplasmic petite strain and the observed 2:2 segregation of the meiotic spore progeny indicate that the mutations are nuclearly inherited and it may be presumed that in each case a nuclear gene product is affected. It is not clear, however, that the mutated proteins are constituent polypeptides of the enzymes in question. For example, it cannot be excluded that some of the cytochrome oxidase mutants which show a total lack of cytochromes a and a1 may be blocked in heme a biosynthesis.
The cytochrome oxidase mutants fall into at least 10 different genetic complementation groups. The mutants can also be distinguished by their spectral properties and patterns of mitochondrial products. For instance, a large number of the strains show a total absence of spectral cytochromes a and a,. Some of the strains in this group may be similar to the nuclear mutants of cytochrome oxidase reported by Sherman and Slonimski (15), &.rbik et al. (16) and Ebner et al. (17). A few mutants in this group have a mitochondrial product of cytochrome oxidase missing as has also been reported by Ebner et al. (17) for their mutants. Another group of cytochrome oxidase mutants have reduced but still detectable amounts of  spectral cytochromes a and u3. These mutants show normal patterns of mitochondrial products. The coenzyme QH,-cytochrome c reductase-specific mutants consist of at least nine complementation groups. These mutants also form different classes which are distinguished by their spectral properties. One group shows a total absence of cytochrome b. These may be similar to the nuclear mutants reported by gubik et al. (18). A second group has reduced levels of the cytochrome and a third exhibits a normal cytochrome spectrum. Two of the mutants (N5-26 and N6-70) have one of the major mitochondrial products missing. Both strains are also devoid of cytochrome b. We do not know at present whether the missing protein is identical to the heme-carrying polypeptide which has been found to be a mitochondrial product in Neurospora (14).
Among the mutants examined, those showing a deficiency in mitochondrial ATPase were the least common. The three that were found were genetically distinct by complementation criteria. Two of the mutants were characterized by an absence of mitochondrial ATPase when this activity was assayed in the mitochondrial and post-ribosomal fractions. These two mutants showed normal patterns of mitochondrial products and exhibited both NADH-cytochrome c reductase and cytochrome oxidase activities, although the latter were considerably lower than in wild type. Since neither strain possesses enzymatically detectable ATPase, it is probable that the mutations affect the synthesis of functional F, ATPase. Ebner and Schatz (19) have previously reported the isolation of a mutant which lacks both enzymatically and immunologically detectable F,. Their mutant also lacked respiratory activities and probably represents yet another class of ATPase mutants.
The third ATPase   (N9-168) was found to be capable of synthesizing F,, but the enzyme did not become integrated into a membrane bound rutamycin-sensitive complex. This mutant shows an interesting pattern of mitochondrial products. The highest molecular weight polypeptide synthesized by mitochondria is completely absent in N9-168. This product is one of the major proteins made in mitochondria and has previously been shown to be a subunit of the rutamycin-sensitive ATPase complex (9). The enzymatic phenotype of N9-168 appears to be similar to some of the ATPase mutants of Schizosaccharomyces pombe reported by Goffeau et al. (20, 21) which also synthesize F, but do not form the rutamycin-sensitive complex. Although this study was primarily concerned with a general characterization of mutants showing specific lesions in the enzymes known to be jointly made by the mitochondrial and cytoribosomal systems of protein synthesis, several interesting observations have emerged. The first is the rather large number of genetic complementation groups that are found within the class of cytochrome oxidase and coenzyme QH2cytochrome c reductase mutants. Since cytochrome oxidase is known to contain only four polypeptides that are synthesized on cytoplasmic ribosomes and coded by nuclear DNA (12), this finding implies the presence of a substantial number of additional nuclear gene products that are necessary for the biosynthesis of the enzyme. A similar conclusion may be drawn about coenzyme QH,-cytochrome c reductase. Ebner et al. (17) recently have described nuclear mutants of cytochrome oxidase in which some of the mitochondrial products of the enzyme fail to be made. In a subsequent study Ono et al. (22) concluded that a nuclear gene product in some of the cytochrome oxidase mutants in some way influenced the synthesis of one of the mitochondrial products. Evidence presented in this study suggests that the control of mitochondrial protein synthesis by nuclear genes may be a more general phenomenon that applies as well to the biosynthesis of coenzyme QH,-cytochrome c reductase and ATPase.