Identification and isolation of the cytochrome oxidase subunit II gene in mitochondria of the yeast Hansenula saturnus.

Mitochondrial DNA from the petite negative yeast Hansenula saturnus has been isolated and sized by digestion with restriction enzymes. The size of the mitochondrial genome is approximately 47 kb. The gene for subunit II of cytochrome oxidase was localized in the genome by Southern blotting using a [32P]-labeled probe containing the subunit II gene of the yeast Saccharomyces cerevisiae. The probe hybridized to a 1.7 kb HindIII-BamHI fragment under stringent conditions (65 degrees C), indicating a high degree of homology between the S. cerevisiae and H. saturnus mitochondrial DNA fragments. The 1.7 kb fragment from H. saturnus was cloned into pBR322 and physically mapped. The map was used to obtain the nucleotide sequence of the subunit II gene (Lawson and Deters presented in the accompanying paper).

Nucleotide sequence of the mitochondrial cytochrome oxidase subunit II gene in the yeast Hansenula saturnus.

The gene for subunit II of cytochrome oxidase in the yeast Hansenula saturnus was previously shown to be located on a 1.7 kb HindIII-BamHI fragment of mitochondrial DNA (Lawson and Deters, accompanying paper). In this paper, we report the nucleotide sequence of a large part of this fragment, covering the coding region of the subunit II gene, designated coxII, and its 5' and 3' flanking regions. The coding region of the coxII gene consists of a continuous open reading frame, 744 nucleotides long, containing 6 in frame TGA codons. Examination of the sequence and alignment with known homologous gene sequences of other organisms indicates that TGA codes for tryptophan in H. saturnus mitochondria as it does in several other mitochondria. Despite considerable homology to subunit II of Saccharomyces cerevisiae, there are 9 codons used in coxII that are not used in the corresponding S. cerevisiae gene. CTT, which is believed to code for threonine in S. cerevisiae mitochondria, appears 3 times in coxII and probably codes for leucine. While the CGN family is rarely, if ever, used in S. cerevisiae mitochondria, CGT appears 4 times in coxII and probably codes for arginine. The deduced amino acid sequence, excluding the first ten amino acids at the N-terminus, is 81% homologous to the amino acid sequence of the S. cerevisiae subunit II protein. The first ten amino acids at the N-terminus are not homologous to the N-terminus of the S. cerevisiae protein but are highly homologous to the first ten amino acids of the deduced amino acid sequence of subunit II of Neurospora crassa. Minor variations of a transcription initiation signal and an end of message or processing signal reported in S. cerevisiae are found in the regions flanking the H. saturnus coxII gene. The subunit II gene contains numerous symmetrical elements, i.e. palindromes, inverted repeats, and direct repeats. Some of these have conserved counterparts in the S. cerevisiae subunit II gene, suggesting that they may be functionally or structurally important.

Identification and characterization of mitochondrial translation products in various yeasts and in Prototheca zopfii.

Mitochondrial genomes of different eucaryotes are not all alike. We have examined mitochondrial translation products in a number of yeasts (Candida krusei, Hansenula saturnus, Rhodotorula glutinis, Rhodotorula rubra, Torulopsis glabrata and Saccharomyces cerevisiae) and in Prototheca zopfii, a colorless alga, in order to determine whether certain proteins are invariably synthesized within mitochondria, how different these proteins are, and what additional proteins, if any, might be synthesized by diverse mitochondria. Using a variety of techniques and criteria, including immunological analysis and peptide mapping, we show that all the yeasts studied, and probably P. zopfii as well, make versions of the 3 large subunits of cytochrome c oxidase. Not all of these oxidase subunits are equally closely related to their counterparts in S. cerevisiae, however. Mitochondria of some of the yeasts studied do not make, or make only small amounts of, a counterpart to Varl, a major mitochondrially made protein in S. cerevisiae. Mitochondria of P. zopfii possibly do not make an apocytochrome b. T. glabrata, H. saturnus and the two Rhodotorula species each make one or more proteins whose relationship, if any, to mitochondrial translation products of S. cerevisiae is not apparent. These results provide new information about mitochondrial diversity. Whereas mitochondria of all the organisms that we have studied devote the major part of their synthetic effort to making the three large subunits of cytochrome c oxidase, and probably make certain other proteins in common, they do not all synthesize a completely identical set of proteins.

Partial resolution of the enzymes catalyzing photophosphorylation. XV. Approaches to the active site of coupling factor I.

1. Prolonged treatment of coupling factor I (CF1) from spinach chloroplasts with trypsin free of chymotrypsin yielded an active ATPase. The isolated preparation showed only two polypeptide chains (mol wt 55,000 to 60,000) on acrylamide gels run in the presence of sodium dodecyl sulfate. The three smaller subunits of CF1 were not detectable. The preparation no longer served as a coupling factor for photophosphorylation in either EDTA- or silicotungstate-treated chloroplasts. 2. An antiserum prepared against coupling factor I from chloroplasts inhibited the ATPase activity of the trypsin-treated CF1. In contrast, antisera prepared against the two individual (denatured) subunits did not inhibit the ATPase activity when tested either alone or together, although each interacted with the trypsin-treated protein, forming precipitin lines in Ouchterlony plates. 3. The trypsin-treated enzyme was still cold-labile, showing that the three smaller subunits are not required for this property. However, the enzyme was no longer sensitive to the natural inhibitor protein which is one of its subunits (subunit epislon), but was still sensitive to inhibition by the flavonoid quercetin. 4. Two equivalents of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole were sufficient to inhibit about 80% of the ATPase activity of the coupling factor, irrespective of whether it contained two of five subunits. The inhibition was completely reversed by dithiothreitol. 5. Triated 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was prepared. Treatment of the coupling factor with this tritium-labeled inhibitor followed by electrophoresis on acrylamide gels revealed that most of the radioactivity was incorporated into the beta subunit of the enzyme (molecular weight 56,000).