Cycles of mitochondrial energization driven by the ultradian clock in a continuous culture of Saccharomyces cerevisiae

Microbiology (BIOSI 1, Main Building), Cardiff University, PO Box 915, Cardiff CF10 3TL, Wales, UK 1 School of Applied Science, University of the South Bank, 103 Borough Road, London SET 0AA, UK 2 Author for correspondence: David Lloyd. Tel: +44 29 20874772. Fax: +44 29 20874305. e-mail: Lloydd{at}cf.ac.uk A continuous culture of Saccharomyces cerevisiae IFO 0233, growing with glucose as the major carbon and energy source, shows oscillations of respiration with a period of 48 min. Samples taken at maxima and minima indicate that (i) periodic changes do not occur as a result of carbon depletion, (ii) intrinsic differences in respiratory activity occur in washed organisms and (iii) a respiratory inhibitor accumulates during respiratory oscillations. Plasma membrane and inner mitochondrial membranes generate transmembrane electrochemical potentials; changes in these can be respectively assessed using anionic or cationic fluorophores. Thus flow cytometric analyses indicated that an oxonol dye [DiBAC 4 (3); bis(1,3-dibutylbarbituric acid)trimethine oxonol] was excluded from yeasts to a similar extent (in >98% of the population) at all stages, showing that the plasma membrane potential was maintained at a steady value. However, uptake of Rhodamine 123 was greatest at that phase characterized by a low respiratory rate. Addition of uncouplers of energy conservation [CCCP ( m -chlorocarbonylcyanide phenylhydrazone) or S-13(5-chloro-3- t -butyl-2-chloro-4 1 -nitrosalicylanilide)] to the continuous cultures increased the respiration, but had only a transient effect on the period of the oscillation. Electron microscopy showed changes in mitochondrial ultrastructure during the respiratory oscillation. At low respiration the cristae were more clearly defined due to swelling of the matrix; this corresponds to the ‘orthodox’ conformation. When respiration was high the mitochondrial configuration was ‘condensed’. It has been shown previously that a temperature-compensated ultradian clock operates in S. cerevisiae . It is proposed that mitochondria undergo cycles of energization in response to energetic demands driven by this ultradian clock output. Keywords: mitochondria, redox regulation, NADH, cytochrome, ultradian clock Abbreviations: CCCP, m -chlorocarbonylcyanide phenylhydrazone; DiBAC 4 (3), bis(1,3-dibutylbarbituric acid)trimethine oxonol; S-13, 5-chloro-3- t -butyl-2-chloro-4 1 -nitrosalicylanilide; DOT, dissolved O 2 tension; SHAM, salicylhydroxamic acid