Anna Maria Viola

A Kluyveromyces lactis gene homologue to AAC2 complements the Saccaromyces cerevisiae op1 mutation

A mutation (op1) in the Saccharomyces cerevisiae AAC2 gene, which codes for the most abundant ADP/ATP carrier isoform, results in lack of mitochondrial-dependent growth and in an as yet unexplained petite-negative phenotype. A gene from the petite-negative yeast Kluyveromyces lactis has been isolated by complementing in multicopy the op1 mutation of S. cerevisiae. This gene, designated KIAAC, can complement the petite-negative phenotype of op1 as well as its inability to grow on non-fermentable carbon sources. KIAAC contains a 915-base pair open reading frame coding for a protein of 305 amino acids which shows a high degree of identity to AAC2. The K. lactis ADP/ATP carrier also shares identity with other known ADP/ATP carrier sequences. In particular, the degree of identity of KIAAC is higher with the Neurospora crassa carrier (80.1%) than with AAC1 (76.6%). The nucleotide sequence upstream of the KIAAC coding region was found to contain a long DNA segment with no coding potential, but presenting features of highly regulated promoter sequences.

Dependence of cytoplasmic on mitochondrial protein synthesis inK. lactis CBS 2360

SummaryIn the “petite negative” yeastKluyveromyces lactis (strain CBS 2360) growth is inhibited by the same Erythromycin concentrations on fermentable and non-fermentable carbon sources. The minimal inhibitory concentration of Erythromycin does not affect respiration, but completely inhibits total aminoacid incorporation by the cells in conditions in which permeability effects can be ruled out.The effect of the antibiotic on the “in vitro” protein synthesis by cytoplasmic and mitochondrial systems shows that inhibition is effective only at the mitochondrial level.Results suggest that the inhibition of growth and of total protein synthesis is a consequence of the inhibition of mitochondrial protein synthesis.

Antimycin A- and hydroxamate-insensitive respiration in yeasts

In this paper evidence is presented for the mitochondrial localization of the antimycin A (AA) + salicylhydroxamate (SHAM)-insensitive respiration of the yeasts Kluyveromyces lactis, Endomycopsis capsularis and Hansenula saturnus. Such a respiration, which can be sustained by NADH and NADPH but not by succinate, is inhibited by high concentrations of azide. AA + SHAM-insensitive respiration is not phosphorylating and its postulated physiological role is to oxidize NADH.

A Klaac null mutant of Kluyveromyces lactis is complemented by a single copy of the Saccharomyces cerevisiae AAC1 gene

Abstract The KlAAC gene, encoding the ADP/ATP carrier in Kluveromyces lactis, has previously been cloned by complementation of the op1(aac2) mutation of Saccharomyces cerevisiae. We examined the effect of a null mutation of this gene on the phenotype of K. lactis. The consequence of this mutation was found to be multiple. The mutant was respiratory deficient, had an undetectable level of cytochrome a-a3 and b and did not grow on glycerol. The mitochondrial D-lactate ferricytochrome c oxidoreductase activity, as well as the lactate-induced transcription of its gene, KlDLD, was severely reduced. Furthermore, the mutant was unable to grow on galactose, maltose and raffinose. Transcript analysis showed that KlAAC was the only ADP/ATP carrier gene present in K. lactis. The Klaac mutation was fully complemented not only by AAC2, the major gene for the ADP/ATP carrier in S. cerevisiae, but also by AAC1, a gene which is poorly expressed in S. cerevisiae. AAC1 introduced in K. lactis was transcribed to a high level consistent with normal growth on glycerol being restored in the transformed mutant. KlAAC was not subject to control by KlHap2, in contrast to AAC2 which is regulated by the Hap2 complex in S. cerevisiae.

The respiratory activities of four Hansenula species

The respiratory activities and the cytochrome spectra from four species belonging to the genus Hansenula have been analysed. The results obtained and described in this paper show that (1) H. glucozyma posseses only the primary, antimycin A-sensitive respiration, (2) H. anomala and H. californica possess primary and secondary (salicylhydroxamate-sensitive) respirations, whereas (3) H. saturnus possesses three respiratory activities (AA-sensitive, SHAM-sensitive, and AA + SHAM-insensitive). The respiratory activity of H. glucozyma is glucose-repressible, whereas the activities of the other species are not. In addition, antimycin A (AA) and erythromycin (ERY) in the culture media differently inhibit the growth of the four species and regulate the respiratory pathways in the species analysed.

Respiratory pathways in Hansenula saturnus.

Hansenula saturnus is a petite-negative yeast species which displays a different pattern of respiration depending on the age of the cultures. The respiration is sensitive to antimycin A (AA) in the early exponential phase, is sensitive to the simultaneous addition of AA and salicylhydroxamic acid (SHAM) in the middle exponential phase and is sensitive to SHAM in the late exponential and stationary phase. The three respiratory activities are all associated to the mitochondrial fraction.The presence of AA in the growth medium determines the induction of the AA+SHAM-insensitive respiration which is 50% inhibited by 5 mM azide. On the contrary, the presence of erythromycin in the growth medium, which inhibits mitochondrial protein synthesis in this yeast species and the synthesis of cytochromes aa3 and b, totally prevents the appearance of AA+SHAM-insensitive respiration. Moreover, the antibiotic affects cell viability, suggesting a role of the mitochondrial protein synthesis in the cell cycle of H. saturnus.

Sporulation and respiratory metabolism in the "petite negative" yeast Hansenula saturnuss.

Upon transition from growth medium to acetate sporulation medium buffered at pH 6.1 with 0.2 M PIPES, Hansenula saturnus showed a respiratory activity which was 88% antimycin A sensitive (1st) and 12% high azide sensitive (3rd), as in acetate complete growth medium. After ≅ 10 h, 3rd respiration declined and oxygen consumption was inhibited by the simultaneous addition of antimycin A and hydroxamate, a situation which lasted until the appearance of the first asci. Later on, 1st and 3rd respiration reappeared and asci formation was completed under these respiratory conditions. The growth in the presence of antimycin A or erythromycin affected only quantitatively the ascospore production and this is because in sporulation medium there was a de novo synthesis of the mitochondrial components of the respiratory chain. Cells which were avoid of 1st respiration but possessed 2nd or 3rd respiration could sporulate, indicating that these alternative respirations also have a role in the process. This was confirmed by the inhibition of sporulation as occurred in the presence of inhibitors of 1st, 2nd and 3rd respiration in sporulation medium.

The in vivo effect of acriflavine on mitochondrial functions in the petite negative yeast Hansenula saturnus

SummaryIn the petite negative yeast Hansenula saturnus, acriflavine determined a decrease of cell yield and of the total QO2,the disappearance of the cytochromes aa3 and b and the inhibition of in vivo mitochondrial protein synthesis without affecting the cell survival. The restriction enzymes analysis of mitDNA shows that no specific fragmentation occurred after acriflavine treatment.