Janet E. Lawson

Cloning, Expression, and Properties of the Regulatory Subunit of Bovine Pyruvate Dehydrogenase Phosphatase

cDNA encoding the regulatory subunit of bovine mitochondrial pyruvate dehydrogenase phosphatase (PDPr) has been cloned. Overlapping cDNA fragments were generated by the polymerase chain reaction from bovine genomic DNA and from cDNA synthesized from bovine poly(A)+ RNA and total RNA. The complete cDNA (2885 base pairs) contains an open reading frame of 2634 nucleotides encoding a putative presequence of 31 amino acid residues and a mature protein of 847 residues with a calculatedM r of 95,656. This value is in agreement with the molecular mass of native PDPr (95,800 ± 200 Da) determined by matrix-assisted laser desorption-ionization mass spectrometry. The mature form of PDPr was expressed inEscherichia coli as a maltose-binding protein fusion, and the recombinant protein was purified to near homogeneity. It exhibited properties characteristic of the native PDPr, including recognition by antibodies against native bovine PDPr, ability to decrease the sensitivity of the catalytic subunit to Mg2+, and reversal of this inhibitory effect by the polyamine spermine. A BLAST search of protein data bases revealed that PDPr is distantly related to the mitochondrial flavoprotein dimethylglycine dehydrogenase, which functions in choline degradation.

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

SummaryMitochondrial 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°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).

Pyruvate dehydrogenase phosphatase

Four classes of protein serine/threonine phosphatases have been identified in eukaryotic cells on the basis of substrate specificities and sensitivity to activators and inhibitors (Cohen, 1989; Shenolikar and Nairn, 1991). Protein phosphatase 1 is sensitive to the thermostable proteins inhibitor 1 and inhibitor 2, and protein phosphatases 1 and 2A are sensitive to okadaic acid. Protein phosphatase 2B is Ca2+/calmodulin-regulated and protein phosphatase 2C is Mg2+-dependent. The protein serine/threonine phosphatases of known sequence comprise two distinct gene families, a major family that includes protein phosphatases 1, 2A, and 2B and isoforms thereof, and a smaller family that includes protein phosphatase 2C and pyruvate dehydrogenase (PDH) phosphatase.

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

SummaryThe 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.