Subunit VII of ubiquinol:cytochrome-c oxidoreductase from Neurospora crassa is functional in yeast and has an N-terminal extension that is not essential for mitochondrial targeting.

cDNA clones encoding subunit VII of the Neurospora crassa bc1 complex (ubiquinol:cytochrome-c oxidoreductase), which is homologous with subunit VIII of the complex from yeast (encoded by QCR8), were identified on the basis of functional complementation of a yeast QCR8 deletion strain. The clones contain an open reading frame encoding a protein with a calculated molecular mass of 11.8 kDa. The N-terminal eight residues of the amino acid sequence deduced from the cDNA clones are absent from the mature protein, as revealed by direct sequencing of the isolated protein. To investigate the potential role of the N-terminal octapeptide in mitochondrial targeting, constructs were made encoding the precursor and the mature form of subunit VII from Neurospora. Incubation of isolated mitochondria with the two proteins revealed that the N-terminal extension of the precursor is removed on import. However, the presequence does not encode information for targeting, as the proteins encoded by both constructs can be imported into isolated mitochondria with equal efficiency. In contrast, the octapeptide seems to have functional importance: the defect in the yeast qcr8-null mutant is not complemented on transformation with the construct encoding mature subunit VII from N. crassa in a single-copy plasmid. We therefore speculate that the N-terminal extension plays a role in intramitochondrial sorting of N. crassa subunit VII. This is supported by the fact that the subunit VII precursor is processed by a protease other than the general mitochondrial processing peptidase. Interestingly, the presequence of N. crassa subunit VII has an amino acid composition similar to the octapeptides cleaved off by the mitochondrial intermediate peptidase.

Carbamoyl phosphate and ureagenesis are not involved in amino-acid-stimulated glycogenesis.

Amino acids are known to stimulate glycogen synthesis via an increase in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959]. It has recently been postulated, however, that carbamoyl phosphate, an intermediate of ureagenesis, can function as a substrate for glucose phosphorylation via carbamoyl-phosphate:glucose phosphotransferase activity of the glucose-6-phosphatase system. This hypothesis would account for the stimulation of glycogenesis by amino acids such as glutamine and proline [Bode, A. M. & Nordlie, R. C. (1993) J. Biol. Chem. 268, 16298-16301]. To further examine the role carbamoyl phosphate may play in glycogenesis, isolated hepatocytes were incubated under a variety of conditions to manipulate ureagenesis, glycogenesis and carbamoyl-phosphate levels. Our data indicate that carbamoyl-phosphate levels do not correlate with amino-acid-stimulated glycogenesis and that ureagenesis and glycogenesis are not competing metabolic pathways.