Dynamic changes in mitochondrial membrane protein composition and proteome redox changes during the yeast metabolic cycle

Bruce Morgan - Frederik Sommer/Michael Schroda - Timo Mühlhaus

Yeast cells grown in continuous culture can synchronize spontaneously and stably with respect to a phenomena termed the yeast metabolic cycle (YMC). The YMC is homologous in many respects to circadian clocks that are widely present in other organisms. The YMC involves coordinated cell cycle division, large-scale synchronized oscillations in transcript levels and oscillations in many cellular metabolites. YMC progression can be observed by monitoring dissolved oxygen levels in continuous culture which exhibit high amplitude oscillations as the cells periodically switch between predominantly fermentative and predominantly respirative modes of metabolism. The YMC is believed to be intrinsic to all yeast cells but is observed in the controlled conditions of a continuous culture due to the population synchronization which is achieved. According to biological dogma, it could be assumed that the coordinated transcriptional and metabolic oscillations in the YMC are simply a result of cell cycle synchrony: cyclin-CDK complexes drive transcriptional changes, which in turn give rise to changes in cellular metabolism. However, recent studies have clearly demonstrated that cell-cycle-associated transcriptional and metabolic cycles can still be observed in the absence of any classical hallmark of cell cycle progression and even proceed in the absence of any mitotic or G1 cyclin. Thus it appears that there are free-running cell-cycle associated transcriptional cycles that are (or can be) independent of cyclin-CDK activity. Likewise recent studies indicate that there are circadian and ultradian (shorter than a day) metabolic/redox cycles that proceed independently of any transcriptional activity. Perturbations in redox state during the YMC impact upon transcriptional and cell cycles suggesting a causal relationship in the opposite direction to what might classically be expected. At minimum, it appears that cellular time-keeping is much more complex than previously assumed and is based upon multiple independent oscillators (metabolic, transcriptional, cyclin-CDK) with extensive cross-talk between them.

In this BioComp project we plan two subprojects. First, during the YMC as the cells switch back and forth between different modes of metabolism, gross changes in mitochondrial morphology have been observed. Mitochondria switch between an 'orthodox' state with tightly folded cristae and a close association between the inner and outer mitochondrial membranes and a 'condensed' state with less folded cristae and greater separation between the inner and outer membranes. It has previously been observed that these changes are associated with mitochondrial activity. However, how the changes are mediated at the protein and membrane level is unclear. In this subproject we will investigate mitochondrial membrane protein composition in mitochondria isolated from cells at different stages of the YMC. This project will contribute to the BioComp consortium in several aspects, for example in the establishment of techniques for obtaining ultra-pure samples and in the development of bioinformatic methods for reducing 'noise' in proteomic mass spectrometry data.

In the second project we will use redox proteomics to look for changes in protein redox states (specifically cysteine thiol oxidation to disulfide bonds) as cells progress through the YMC. We observe that perturbations of redox state, especially the addition of peroxides to YMC-synchronized yeast cultures, strongly perturbs YMC progression, in a manner that is dependent upon the phase of the YMC at which peroxide is added. At some defined phases of the YMC, peroxide addition can even trigger a complete resetting of the YMC leading to initiation of a new YMC cycle. Interestingly, we observe changes in endogenous peroxide levels during the YMC that lead to changes in the oxidation state of thiol peroxidases. Deletion of thiol peroxidases (peroxide scavenging enzymes) can modulate the effects of peroxide addition. We speculate that thiol peroxidases pass on their oxidation to specific target protein, for example transcription factors, thereby serving to couple metabolic and transcriptional cycles. We are currently using OxICAT methodology to observe changes in protein redox state during the YMC and following treatment with exogenous peroxide. We will investigate how these changes are affected by the deletion of specific thiol peroxidases either alone or in combination.