It has recently been shown that cyanide-resistant respiration (CRR) is very common in Crabtree-negative yeasts (incapable of aerobic fermentation) and in non-fermentative yeasts. It is conferred by a salicylhydroxamic acid-sensitive alternative oxidase that transfers electrons from ubiquinol to oxygen, bypassing the cytochrome chain. An interesting finding is that, in general, whenever CRR is present, complex I is also present. In this article we briefly review the occurrence of CRR, the biochemistry and molecular biology of the alternative oxidase, and summarise the putative functions that have been attributed to this ubiquitous metabolic pathway, whose usefulness for the yeast cells still remains obscure.

Aims: To investigate the conditions that promote the expression of cyanide-resistant respiration (CRR) in the spoilage yeasts Pichia membranifaciens and Debaryomyces hansenii. Methods and Results: CRR was detected by sensitivity of oxygen consumption to salicylhydroxamic acid. It was absent in both yeasts in the early exponential phase, but was triggered by several stress situations. Starvation under aerobic conditions, decreasing pH or incubation of the culture in a narrow temperature range below the maximum temperature for growth promoted the emergence of CRR in both yeasts. In D. hansenii, CRR was also induced by 1AE5-2 mol l )1 NaCl. Although the presence of H 2 O 2 and menadione induced CRR, radical scavengers had no effect on the emergence of CRR. Also, the level of reactive oxygen species did not vary with the CRR activity.Conclusions: Under aerobic conditions, a respiratory pathway alternative to the cytochrome chain is triggered by stress conditions in P. membranifaciens and D. hansenii. Significance and Impact of the Study: The relationship between stress situations and CRR must be taken into account in studies on the performance of spoilage yeasts in the food processing environments where several forms of stress are common.

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (Delta Psi(m)), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.

In Pichia membranifaciens, cyanide-resistant respiration (CRR) sensitive to salicylhydroxamic acid emerged after forced aeration of starved cells for 4 h. Surveying a large number of species by this simple methodology, we found that CRR is very frequent among yeasts. Remarkably, considering our results together with previous data in the literature, CRR was present in 24 out of 28 non-fermentative or Crabtree-negative yeasts and absent in 10 out of 12 Crabtree-positive yeasts. We submit that, as alternatives to cytochromic respiration, yeasts developed two strategies: either aerobic fermentation in Crabtree-positive yeasts or CRR in non-fermentative or Crabtree-negative yeasts. ß

The importance of aquaporin expression in water permeability in Saccharomyces cerevisiae was assessed by measuring the osmotic water permeability coefficient (P f ) and the activation energies (E a ) from both hypo-and hypertonic experiments performed with whole protoplasts from four strains differing in aquaporin level of expression: parental, double-deleted and overexpressing AQY1 or AQY2. Double-deleted (lower P f ) and AQY1-overexpressing strains (higher P f ) presented linear Arrhenius plots with E a consistent with fluxes mainly through the lipids [16?3 kcal mol "1 (68?2 kJ mol )], respectively. The Arrhenius plots for the parental (swelling experiments) and overexpressing AQY2 strains (swelling and shrinking experiments) were not linear, presenting a break point with a change in slope around 23 6C. The E a values for these strains, calculated for temperatures ranging from 7 to 23 6C, were lower [9?5 kcal mol "1 (39?7 kJ mol The permeabilities for each strain relative to the deletion strain show that an increase in permeability due to the presence of aquaporins was more relevant at low temperatures. Following our results, we propose that water channels play an important role for osmotic adjustment of yeast cells at low temperature.