Isolation and properties of promitochondria from anaerobic stationary-phase yeast cells.
Under anaerobiosis, the mitochondrion of Saccharomyces cerevisiae is restricted to unstructured promitochondria. These promitochondria provide unknown metabolic functions that are required for growth. Since high glucose concentrations are mainly fermented by S. cerevisiae during stationary phase (due to nitrogen starvation), an optimized promitochondria isolation procedure was investigated. Firstly, the unusual promitochondria ultrastructure was checked in intact cells by electron microscopy using a cryo-fixation and freeze-substitution method. The rapid response of anaerobic cells toward oxygen justified the adoption of several critical steps, especially during spheroplasting. Control of spheroplasting was accompanied by a systematic analysis of spheroplast integrity, which greatly influence the final quality of promitochondria. Despite the presence of remnant respiratory chain components under anaerobiosis, characterization of isolated promitochondria by high-resolution respirometry did not reveal any antimycin A- and myxothiazol-sensitive NADH and NADPH oxidase activities. Moreover, the existence of a cyanide-sensitive and non-phosphorylating NADH-dependent oxygen consumption in promitochondria was demonstrated. Nevertheless, promitochondria only slightly contribute to the overall oxygen consumption capacity observed in highly glucose-repressed anaerobic cells.
Rosenfeld
Eric
E
Unité Mixte de Recherches Sciences pour l'oenologie, INRA-IPV, 2 place Viala, F-34060 Montpellier Cedex 1, France.
Schaeffer
Jacques
J
Beauvoit
Bertrand
B
Salmon
Jean-Michel
JM
D000332:Aerobiosis; D002458:Cell Fractionation / Q000379:methods; D003470:Culture Media; D005285:Fermentation; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D008353:Mannitol / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000648:ultrastructure*; D009584:Nitrogen / Q000378:metabolism; D010101:Oxygen Consumption; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D013104:Spheroplasts / Q000187:drug effects / Q000502:physiology
D003470:Culture Media; D008353:Mannitol; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D005947:Glucose; D009584:Nitrogen