BACKGROUND
depending on the environmental conditions, and in particular the nature of carbon substrates, saccharomyces cerevisiae adjusts its energy metabolism in a process that is generally referred as carbon catabolite repression.
two extreme cases are exponential growth on glucose and on ethanol, which lead to almost exclusive fermentation with extensive secretion of ethanol and exclusive respiration to carbon dioxide, respectively.
both, fully respiratory and fermentative energy production is mediated, to a large extent, by differentially active transcription factors.
during fermentative metabolism, mainly the transcription factor complex of tup1p, ssn6p and mig1p repress the expression of respiratory, gluconeogenic and alternative carbon source utilization genes  <cit> .
minimal activity of the tricarboxylic acid  cycle for biosynthetic purposes under fermentative conditions is mainly assured by the rtg transcriptional activators  <cit> .
during respiratory growth on non fermentable carbon sources, on the other hand, respiratory genes in the tca cycle and respiratory chain are highly induced.
this is triggered by the hap transcription factors, a global activator complex of respiratory genes  <cit> .
activation of genes required for gluconeogenesis during growth on non fermentable carbon sources is achieved by the transcriptional activators cat8p and sip4p  <cit> .
these are the key elements of the yeast transcriptional regulatory network for the extreme cases of fermentative and fully respirative growth.
however, yeast also consumes carbon sources such as galactose, which is presumed to cause simultaneous respiration and fermentation  <cit> .
another case is mannose, where levels of the typical carbon catabolite repression reporter suc2p show that carbon catabolite repression is significantly decreased  <cit> , which would suggest higher respiration than on glucose.
how is respiration regulated transcriptionally on these carbon sources?
here, we address this question quantitatively by linking the functional degree of respiration to transcriptional regulation via enzyme abundances.
for this purpose, we used galactose and mannose as examples of potential intermediate respiration, and glucose and pyruvate as extreme cases of minimal and maximal respiration in aerobic batch cultures, respectively.
respiration is quantified as the flux through the tca cycle by 13c flux analysis  <cit> .
protein abundances of respiratory enzymes are quantified with gfp fusion strains  <cit> .
from these data we then predicted the most probable transcription factors that control the coordinated differential expression of respiratory enzymes under the various conditions.
these predictions were then verified on the basis of functional changes in the corresponding transcription factor deletion strains, leading to a carbon source dependent model of transcriptional regulation of respiration.
RESULTS
degree of respiration on different carbon sources
to elucidate the transcriptional regulation of respiration, we grew wild-type s. cerevisiae fy <dig> in aerobic microscale batch cultures  on minimal medium with either glucose, mannose, galactose or pyruvate as sole carbon source.
for a functional quantification of respiration, we determined intracellular carbon fluxes with 13c flux analysis in eight independent cultures per carbon substrate  <cit> .
for this purpose, yeast was grown on either 13c labeled glucose, mannose, galactose or pyruvate.
these carbon substrates are degraded via different metabolic pathways that lead to different 13c labeling patterns which were subsequently measured in protein-bound amino acids by gas chromatography - mass spectrometry .
from the determined mass isotopomer abundances in amino acids, we calculated eight ratios of converging central metabolic fluxes with algebraic equations  <cit> .
these determined flux ratios  were then combined with physiological data  such as the carbon uptake rate and a stoichiometric model of central yeast metabolism to fit metabolic fluxes in mmol/g/h to the measured data  <cit>  .
standard deviations were fitted from eight independent cultures.
error ranges for the degree of respiration were fitted with the input of eight independent cultures.
since the overall rate of metabolism differed widely, as can be seen from the substrate uptake rates , we normalized the calculated metabolic fluxes by the uptake rate to enable a direct comparison .
the thus normalized fluxes on glucose and mannose were very similarly distributed.
on galactose, by-product formation was significantly reduced, while the tca cycle and biomass fluxes were increased.
on pyruvate, yeast exhibited exclusively respiratory metabolism without ethanol formation and, of course, a completely reversed glycolytic flux.
in the following we are primarily interested in a quantitative measure of respiration that would be directly comparable between the conditions.
hence we used the normalized flux from mitochondrial malate to mitochondrial oxaloacetate as a measure for the respiratory flux through the tca cycle, which we therefore define as the degree of respiration.
this degree of respiration was marginal on glucose and mannose, significant on galactose and, as expected, highest on pyruvate where energy production completely relies on respiration.
the determined biomass yields and fluxes to ethanol correlated with the degree of respiration, thus confirming the conclusion on the degree of respiratory metabolism of the four carbon sources .
our finding on the different metabolic states during growth on the tested carbon sources are also in agreement with previous studies such as the work of polakis and bartley  <cit> .
protein abundances
the general expectation is that altered levels of respiration are accomplished through modulations in enzyme abundances  <cit> .
to elucidate which of the approximately  <dig> respiratory enzymes are relevant for the different degrees of respiration, we used protein-gfp fusion strains that are typically used in protein localization studies  <cit> .
to use the fluorescence signal of c-terminally tagged proteins to quantify protein abundances raises the issue whether the fused proteins are still catalytically active, e.g.
correctly folded, correctly aggregating into multimers/complexes and still subject of allosteric regulation.
while the final proof is not possible, we provide two lines of evidence that the gfp-fusion proteins function normal in the majority of the cases.
firstly, we quantified the specific growth rate as an overall measure of physiological function.
on average, 80% of the investigated strains grew at a rate that was indistinguishable of the reference strain under the four tested conditions.
secondly, as a more sensitive and specific measure of functionality, we quantified intracellular concentrations of  <dig> metabolites in four gfp-fusion strains , cox8-gfp::his <dig>  qcr6-gfp::his3) and the reference strain during growth on galactose.
we found on average a differences of  <dig>  fold changes  between the metabolite concentrations of fusion and reference strain; only very few metabolites were altered by up to  <dig> fold changes .
collectively, this data indicate that the majority of the gfp-fused proteins are still functional as it was also found by newman et.
al  <cit> .
we investigated in total  <dig> protein-gfp fusion strains, each strain containing one protein-gfp fusion construct.
these include  <dig> enzymes from the tca cycle,  <dig> from the respiratory chain and  <dig> enzymes from the ethanol production pathway .
the coverage for tca cycle, respiratory chain and ethanol production enzymes was more than 84%, 70%  and 78%, respectively.
the  <dig> gfp-tagged strains were grown on the four carbon sources and we determined the steady state enzyme abundances on each of the four carbon sources from online fluorescence and biomass measurements in 96-well plates.
more than 60% of all investigated enzymes changed their level of expression during growth on at least one carbon source compared to glucose .
while the expression levels on mannose were almost identical to those on glucose,  <dig> of  <dig> investigated enzymes that catalyze tca cycle and the respiratory chain reactions were up-regulated on galactose and/or pyruvate and only five enzymes were down-regulated.
the gfp-fusion strains of rip <dig> and cox <dig> did not grow on galactose and pyruvate, very likely because the fusion enzymes were not functional anymore.
thus, rip1p and cox12p were considered as important for respiration.
next we looked for patterns of enzyme expression that are related to respiration and whether these would be over-represented in one of the enzyme categories that we investigated .
there was clearly an over representation of tca cycle enzymes  in enzyme expression patterns that were positively related with respiration .
of all respiratory chain enzymes in contrast only 42% were found to be positively related with respiration .
for each reaction in the tca cycle there was at least one catalyzing enzyme up-regulated under respiratory metabolism with the exception of succinyl-coa ligase for galactose.
for the aconitase catalyzed reaction we had no information about the main enzyme aco1p since it was not available as gfp-fusion.
the isoenzyme aco2p was negatively related with respiration, as expected, since it is induced on glucose and repressed on non-fermentable carbon sources  <cit> .
in the respiratory chain there was on galactose and pyruvate at least one subunit per complex  up-regulated, while for the nadh dehydrogenase only the internal  but not the external one  was increased in expression.
fold changes in enzymes expressed on mannose , galactose  or pyruvate  compared to growth on glucose  .
expression levels are determined during exponential growth in batch with strains containing a single enzyme-gfp construct.
significant changes were assigned when the p-value was ≤  <dig>   compared to the enzyme expression during growth on glucose.
to elucidate a potential quantitative correlation between enzyme levels and degree of respiration, we plotted the measured abundance for each enzyme against the corresponding degree of respiration and calculated the correlation factor .
six respiratory enzyme level - degree of respiration curves exhibited a correlation factor of  <dig>  or higher , additionally correlated pdc1p, an enzyme from the ethanol production pathway, negatively with the degree of respiration.
these findings are consistent with increased in vitro enzyme activities of idh1/2p, fum1p and mdh1p on galactose and pyruvate compared to glucose  <cit> .
in our data set again, enzymes of the tca cycle were over represented in the set of respiratory enzymes that correlate with the degree of respiration.
we thus conclude that mainly the tca cycle is important for establishing the different degrees of respiration under the tested conditions.
while the respiratory chain is certainly also required, all of its expression levels except of those of cor1p do not correlate with the degree of respiration and we have no evidence for a carbon source dependent coordinated up-regulation of all subunits of the respiratory chain.
transcriptional regulation of respiration
since transcriptional regulation is the most probable mechanism to achieve these distinct modulations in respiratory enzyme abundances, we were interested to identify the involved transcription factors.
for this purpose we applied a statistical framework that evaluates the probability of each transcription factor to be responsible for the observed differential expression pattern.
specifically, we used the pairwise fold changes between enzyme levels on galactose and pyruvate compared to those on glucose; i.e.
<dig> and  <dig> enzymes were up-regulated on galactose and pyruvate compared to glucose, respectively .
from the yeastract database  <cit> , we then selected all transcription factors that had at least one target  among the  <dig> investigated enzymes.
our statistical analysis is based on the hypothesis that a transcription factor with the same target genes as the observed pattern of differential expressed enzymes has a high probability to be responsible for causing the change .
transcription factors that include the differentially expressed enzymes in their target pattern, but have many additional targets that were not differentially expressed in our data set, in contrast, have a low probability to be responsible.
specifically, we calculated with a hypergeometric distribution a probability value f, that the target pattern of a transcription factor by chance includes the pattern of measured differential expressed enzymes  <cit>  .
at low probability values f, the considered transcription factor is likely to be involved in the regulation of the observed differential expressed enzymes.
since the biological meaning of the absolute value of f is limited, we considered always the transcription factors with the five lowest f values as relevant.
on galactose compared to glucose, we identified the transcription factors gcr1p, hap2p, hap3p, hap5p, rtg1p and rtg2p as most likely to be responsible for the observed up-regulated enzymes .
on pyruvate compared to glucose, we identified the transcription factors cat8p, gcr1p, hap2p, rtg1p and rtg2p .
for the hap-complex, this result was expected since it is a global activator complex of respiration  <cit> .
the rtg-proteins are only known as activators of some tca cycle genes in cells with compromised mitochondrial function  <cit> , beside their function in retrograde signaling.
cat8p activates the gluconeogenic and glyoxylate cycle genes pck <dig>  fbp <dig>  icl <dig> and mls <dig>  <cit> .
furthermore, it was shown that hap <dig> activation depends on an intact cat <dig> gene  <cit> .
the main known function of gcr1p is the activation of glycolysis genes  <cit> .
from these results we hypothesize that the above transcription factors influence respiration through activation  or repression  of their respiratory target genes.
to verify this hypothesis, we grew deletion mutants of the predicted hap, rtg <dig> and cat <dig> transcription factors on galactose and pyruvate.
we determined the biomass yield and the normalized respiratory tca cycle flux by 13c flux analysis as functional measures of the degree of respiration .
when a predicted transcription factor is indeed responsible for the level of respiration observed in the wild-type strain on galactose or on pyruvate, the degree of respiration and the biomass yields should be decreased in the corresponding deletion mutant.
biomass yield standard deviations were calculated from at least two independent cultures.
error ranges for the degree of respiration were fitted with the input of at least two independent cultures.
the cat <dig> deletion strain did not grow on pyruvate as expected, because cat8p is essential for the activation of gluconeogenic genes  <cit> .
on galactose, where gluconeogenesis is not required the cat <dig> deletion strain exhibited a significantly decreased biomass yield and degree of respiration compared to the wild-type, confirming its importance for respiration.
yet, cat8p is not essential for respiration, since respiration is not completely absent in the cat <dig> deletion strain on galactose .
the importance of cat8p for respiration is thus independent from its function in gluconeogenesis.
this might be explained by a requirement of cat8p for full hap4p activation or the necessity of cat8p activation of its tca cycle targets idh <dig> and fum <dig>  <cit> .
for all hap deletion strains, the degree of respiration and the biomass yield were indeed significantly lower than in the wild-type strain during growth on the partly respired substrate galactose .
on the non fermentable substrate pyruvate, the hap deletion strains were not able to grow.
the respiratory phenotype of the rtg <dig> deletion strain on galactose was basically identical to those of the hap deletion strains .
on pyruvate, in contrast, the rtg <dig> deletion strain exhibited basically a wild-type like biomass yield and degree of respiration.
while the finding for rtg <dig> deletion strain grown on pyruvate fits literature data and models  <cit> , our finding for rtg <dig> deletion strain grown on galactose was unexpected because the literature model  <cit>  ascribes rtg1p activity to impaired mitochondrial function as in  cells, which is certainly not the case for growth on galactose.
CONCLUSIONS
we quantified the proportion of respiratory energy generation on four differentially repressive carbon sources and identified, by combined experimental and computational analysis, key transcription factors that are important for respiration in s. cerevisiae.
we confirmed in this study the essential role of hap2/3/5p in respiration, identified essentiality of rtg1p for respiration on galactose and identified an important, but not essential role for cat8p in attaining a wild-type like degree of respiration.
this cat8p-based regulation of respiration is independent of the known regulation of gluconeogenesis because the cat <dig> mutant grows well on the glycolytic substrate galactose, but with significantly reduced respiration.
while importance of the hap-complex for respiration was known  <cit> , the impact of the rtg-proteins on respiration cannot be solely explained by the previously proposed model that ascribes their activity to cells with highly impaired mitochondrial function  <cit>  or by their stress induced role in retrograde signaling  <cit> .
thus, we propose an expanded model which adds the aspect of carbon source, and thus the degree of respiration dependent activity pattern, for the rtg-proteins and the hap-complex .
the newly added aspect of the carbon source is even seen for the activity pattern of the hap-complex in  cells with highly impaired mitochondrial function during growth on either glucose or raffinose .
this study provides further knowledge on the regulation of respiration on four differentially repressive carbon sources by combining the transcription factor target network with experimentally measured protein expression levels using a statistical framework.

 depending on the environmental conditions, and in particular the nature of carbon substrates, saccharomyces cerevisiae adjusts its energy metabolism in a process that is generally referred as carbon catabolite repression. two extreme cases are exponential growth on glucose and on ethanol, which lead to almost exclusive fermentation with extensive secretion of ethanol and exclusive respiration to carbon dioxide, respectively. both, fully respiratory and fermentative energy production is mediated, to a large extent, by differentially active transcription factors. during fermentative metabolism, mainly the transcription factor complex of tup1p, ssn6p and mig1p repress the expression of respiratory, gluconeogenic and alternative carbon source utilization genes. minimal activity of the tricarboxylic acid cycle for biosynthetic purposes under fermentative conditions is mainly assured by the rtg transcriptional activators. during respiratory growth on non fermentable carbon sources, on the other hand, respiratory genes in the tca cycle and respiratory chain are highly induced. this is triggered by the hap transcription factors, a global activator complex of respiratory genes. activation of genes required for gluconeogenesis during growth on non fermentable carbon sources is achieved by the transcriptional activators cat8p and sip4p. these are the key elements of the yeast transcriptional regulatory network for the extreme cases of fermentative and fully respirative growth. however, yeast also consumes carbon sources such as galactose, which is presumed to cause simultaneous respiration and fermentation. another case is mannose, where levels of the typical carbon catabolite repression reporter suc2p show that carbon catabolite repression is significantly decreased, which would suggest higher respiration than on glucose. how is respiration regulated transcriptionally on these carbon sources? here, we address this question quantitatively by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. for this purpose, we used galactose and mannose as examples of potential intermediate respiration, and glucose and pyruvate as extreme cases of minimal and maximal respiration in aerobic batch cultures, respectively. respiration is quantified as the flux through the tca cycle by 13c flux analysis. protein abundances of respiratory enzymes are quantified with gfp fusion strains. from these data we then predicted the most probable transcription factors that control the coordinated differential expression of respiratory enzymes under the various conditions. these predictions were then verified on the basis of functional changes in the corresponding transcription factor deletion strains, leading to a carbon source dependent model of transcriptional regulation of respiration.  degree of respiration on different carbon sources to elucidate the transcriptional regulation of respiration, we grew wild-type s. cerevisiae fy in aerobic microscale batch cultures on minimal medium with either glucose, mannose, galactose or pyruvate as sole carbon source. for a functional quantification of respiration, we determined intracellular carbon fluxes with 13c flux analysis in eight independent cultures per carbon substrate. for this purpose, yeast was grown on either 13c labeled glucose, mannose, galactose or pyruvate. these carbon substrates are degraded via different metabolic pathways that lead to different 13c labeling patterns which were subsequently measured in protein-bound amino acids by gas chromatography - mass spectrometry. from the determined mass isotopomer abundances in amino acids, we calculated eight ratios of converging central metabolic fluxes with algebraic equations. these determined flux ratios were then combined with physiological data such as the carbon uptake rate and a stoichiometric model of central yeast metabolism to fit metabolic fluxes in mmol/g/h to the measured data. standard deviations were fitted from eight independent cultures. error ranges for the degree of respiration were fitted with the input of eight independent cultures. since the overall rate of metabolism differed widely, as can be seen from the substrate uptake rates, we normalized the calculated metabolic fluxes by the uptake rate to enable a direct comparison. the thus normalized fluxes on glucose and mannose were very similarly distributed. on galactose, by-product formation was significantly reduced, while the tca cycle and biomass fluxes were increased. on pyruvate, yeast exhibited exclusively respiratory metabolism without ethanol formation and, of course, a completely reversed glycolytic flux. in the following we are primarily interested in a quantitative measure of respiration that would be directly comparable between the conditions. hence we used the normalized flux from mitochondrial malate to mitochondrial oxaloacetate as a measure for the respiratory flux through the tca cycle, which we therefore define as the degree of respiration. this degree of respiration was marginal on glucose and mannose, significant on galactose and, as expected, highest on pyruvate where energy production completely relies on respiration. the determined biomass yields and fluxes to ethanol correlated with the degree of respiration, thus confirming the conclusion on the degree of respiratory metabolism of the four carbon sources. our finding on the different metabolic states during growth on the tested carbon sources are also in agreement with previous studies such as the work of polakis and bartley. protein abundances the general expectation is that altered levels of respiration are accomplished through modulations in enzyme abundances. to elucidate which of the approximately respiratory enzymes are relevant for the different degrees of respiration, we used protein-gfp fusion strains that are typically used in protein localization studies. to use the fluorescence signal of c-terminally tagged proteins to quantify protein abundances raises the issue whether the fused proteins are still catalytically active, e.g. correctly folded, correctly aggregating into multimers/complexes and still subject of allosteric regulation. while the final proof is not possible, we provide two lines of evidence that the gfp-fusion proteins function normal in the majority of the cases. firstly, we quantified the specific growth rate as an overall measure of physiological function. on average, 80% of the investigated strains grew at a rate that was indistinguishable of the reference strain under the four tested conditions. secondly, as a more sensitive and specific measure of functionality, we quantified intracellular concentrations of metabolites in four gfp-fusion strains, cox8-gfp::his qcr6-gfp::his3) and the reference strain during growth on galactose. we found on average a differences of fold changes between the metabolite concentrations of fusion and reference strain; only very few metabolites were altered by up to fold changes. collectively, this data indicate that the majority of the gfp-fused proteins are still functional as it was also found by newman et. al. we investigated in total protein-gfp fusion strains, each strain containing one protein-gfp fusion construct. these include enzymes from the tca cycle, from the respiratory chain and enzymes from the ethanol production pathway. the coverage for tca cycle, respiratory chain and ethanol production enzymes was more than 84%, 70% and 78%, respectively. the gfp-tagged strains were grown on the four carbon sources and we determined the steady state enzyme abundances on each of the four carbon sources from online fluorescence and biomass measurements in 96-well plates. more than 60% of all investigated enzymes changed their level of expression during growth on at least one carbon source compared to glucose. while the expression levels on mannose were almost identical to those on glucose, of investigated enzymes that catalyze tca cycle and the respiratory chain reactions were up-regulated on galactose and/or pyruvate and only five enzymes were down-regulated. the gfp-fusion strains of rip and cox did not grow on galactose and pyruvate, very likely because the fusion enzymes were not functional anymore. thus, rip1p and cox12p were considered as important for respiration. next we looked for patterns of enzyme expression that are related to respiration and whether these would be over-represented in one of the enzyme categories that we investigated. there was clearly an over representation of tca cycle enzymes in enzyme expression patterns that were positively related with respiration. of all respiratory chain enzymes in contrast only 42% were found to be positively related with respiration. for each reaction in the tca cycle there was at least one catalyzing enzyme up-regulated under respiratory metabolism with the exception of succinyl-coa ligase for galactose. for the aconitase catalyzed reaction we had no information about the main enzyme aco1p since it was not available as gfp-fusion. the isoenzyme aco2p was negatively related with respiration, as expected, since it is induced on glucose and repressed on non-fermentable carbon sources. in the respiratory chain there was on galactose and pyruvate at least one subunit per complex up-regulated, while for the nadh dehydrogenase only the internal but not the external one was increased in expression. fold changes in enzymes expressed on mannose, galactose or pyruvate compared to growth on glucose. expression levels are determined during exponential growth in batch with strains containing a single enzyme-gfp construct. significant changes were assigned when the p-value was ≤ compared to the enzyme expression during growth on glucose. to elucidate a potential quantitative correlation between enzyme levels and degree of respiration, we plotted the measured abundance for each enzyme against the corresponding degree of respiration and calculated the correlation factor. six respiratory enzyme level - degree of respiration curves exhibited a correlation factor of or higher, additionally correlated pdc1p, an enzyme from the ethanol production pathway, negatively with the degree of respiration. these findings are consistent with increased in vitro enzyme activities of idh1/2p, fum1p and mdh1p on galactose and pyruvate compared to glucose. in our data set again, enzymes of the tca cycle were over represented in the set of respiratory enzymes that correlate with the degree of respiration. we thus conclude that mainly the tca cycle is important for establishing the different degrees of respiration under the tested conditions. while the respiratory chain is certainly also required, all of its expression levels except of those of cor1p do not correlate with the degree of respiration and we have no evidence for a carbon source dependent coordinated up-regulation of all subunits of the respiratory chain. transcriptional regulation of respiration since transcriptional regulation is the most probable mechanism to achieve these distinct modulations in respiratory enzyme abundances, we were interested to identify the involved transcription factors. for this purpose we applied a statistical framework that evaluates the probability of each transcription factor to be responsible for the observed differential expression pattern. specifically, we used the pairwise fold changes between enzyme levels on galactose and pyruvate compared to those on glucose; i.e. and enzymes were up-regulated on galactose and pyruvate compared to glucose, respectively. from the yeastract database, we then selected all transcription factors that had at least one target among the investigated enzymes. our statistical analysis is based on the hypothesis that a transcription factor with the same target genes as the observed pattern of differential expressed enzymes has a high probability to be responsible for causing the change. transcription factors that include the differentially expressed enzymes in their target pattern, but have many additional targets that were not differentially expressed in our data set, in contrast, have a low probability to be responsible. specifically, we calculated with a hypergeometric distribution a probability value f, that the target pattern of a transcription factor by chance includes the pattern of measured differential expressed enzymes. at low probability values f, the considered transcription factor is likely to be involved in the regulation of the observed differential expressed enzymes. since the biological meaning of the absolute value of f is limited, we considered always the transcription factors with the five lowest f values as relevant. on galactose compared to glucose, we identified the transcription factors gcr1p, hap2p, hap3p, hap5p, rtg1p and rtg2p as most likely to be responsible for the observed up-regulated enzymes. on pyruvate compared to glucose, we identified the transcription factors cat8p, gcr1p, hap2p, rtg1p and rtg2p. for the hap-complex, this result was expected since it is a global activator complex of respiration. the rtg-proteins are only known as activators of some tca cycle genes in cells with compromised mitochondrial function, beside their function in retrograde signaling. cat8p activates the gluconeogenic and glyoxylate cycle genes pck fbp icl and mls. furthermore, it was shown that hap activation depends on an intact cat gene. the main known function of gcr1p is the activation of glycolysis genes. from these results we hypothesize that the above transcription factors influence respiration through activation or repression of their respiratory target genes. to verify this hypothesis, we grew deletion mutants of the predicted hap, rtg and cat transcription factors on galactose and pyruvate. we determined the biomass yield and the normalized respiratory tca cycle flux by 13c flux analysis as functional measures of the degree of respiration. when a predicted transcription factor is indeed responsible for the level of respiration observed in the wild-type strain on galactose or on pyruvate, the degree of respiration and the biomass yields should be decreased in the corresponding deletion mutant. biomass yield standard deviations were calculated from at least two independent cultures. error ranges for the degree of respiration were fitted with the input of at least two independent cultures. the cat deletion strain did not grow on pyruvate as expected, because cat8p is essential for the activation of gluconeogenic genes. on galactose, where gluconeogenesis is not required the cat deletion strain exhibited a significantly decreased biomass yield and degree of respiration compared to the wild-type, confirming its importance for respiration. yet, cat8p is not essential for respiration, since respiration is not completely absent in the cat deletion strain on galactose. the importance of cat8p for respiration is thus independent from its function in gluconeogenesis. this might be explained by a requirement of cat8p for full hap4p activation or the necessity of cat8p activation of its tca cycle targets idh and fum. for all hap deletion strains, the degree of respiration and the biomass yield were indeed significantly lower than in the wild-type strain during growth on the partly respired substrate galactose. on the non fermentable substrate pyruvate, the hap deletion strains were not able to grow. the respiratory phenotype of the rtg deletion strain on galactose was basically identical to those of the hap deletion strains. on pyruvate, in contrast, the rtg deletion strain exhibited basically a wild-type like biomass yield and degree of respiration. while the finding for rtg deletion strain grown on pyruvate fits literature data and models, our finding for rtg deletion strain grown on galactose was unexpected because the literature model ascribes rtg1p activity to impaired mitochondrial function as in cells, which is certainly not the case for growth on galactose.  we quantified the proportion of respiratory energy generation on four differentially repressive carbon sources and identified, by combined experimental and computational analysis, key transcription factors that are important for respiration in s. cerevisiae. we confirmed in this study the essential role of hap2/3/5p in respiration, identified essentiality of rtg1p for respiration on galactose and identified an important, but not essential role for cat8p in attaining a wild-type like degree of respiration. this cat8p-based regulation of respiration is independent of the known regulation of gluconeogenesis because the cat mutant grows well on the glycolytic substrate galactose, but with significantly reduced respiration. while importance of the hap-complex for respiration was known, the impact of the rtg-proteins on respiration cannot be solely explained by the previously proposed model that ascribes their activity to cells with highly impaired mitochondrial function or by their stress induced role in retrograde signaling. thus, we propose an expanded model which adds the aspect of carbon source, and thus the degree of respiration dependent activity pattern, for the rtg-proteins and the hap-complex. the newly added aspect of the carbon source is even seen for the activity pattern of the hap-complex in cells with highly impaired mitochondrial function during growth on either glucose or raffinose. this study provides further knowledge on the regulation of respiration on four differentially repressive carbon sources by combining the transcription factor target network with experimentally measured protein expression levels using a statistical framework. 
from these enzyme levels and the known regulatory network structure, we determined the probability for a given transcription factor to cause the coordinated expression changes. the most probable transcription factors to regulate the different degrees of respiration were gcr1p, cat8p, the rtg-proteins and the hap-complex. we address this question by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. during growth on the partly and fully respired substrates galactose and pyruvate, several tca cycle and respiratory chain enzymes were significantly up-regulated. based on 13c flux analysis, we found that the respiratory contribution to cellular energy production was largely absent on glucose and mannose, intermediate on galactose and highest on pyruvate. specifically, we investigated aerobic batch cultures with the differently repressive carbon sources glucose, mannose, galactose and pyruvate. while many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration. depending on the carbon source, saccharomyces cerevisiae displays various degrees of respiration. for the latter three ones we confirmed their importance for respiration by quantifying the degree of respiration and biomass yields in the corresponding deletion strains.
 depending on the carbon source, saccharomyces cerevisiae displays various degrees of respiration. these range from complete respiration as in the case of ethanol, to almost complete fermentation, and thus very low degrees of respiration on glucose. while many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration.  we address this question by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. specifically, we investigated aerobic batch cultures with the differently repressive carbon sources glucose, mannose, galactose and pyruvate. based on 13c flux analysis, we found that the respiratory contribution to cellular energy production was largely absent on glucose and mannose, intermediate on galactose and highest on pyruvate. in vivo abundances of respiratory enzymes were quantified by gfp-fusions under each condition. during growth on the partly and fully respired substrates galactose and pyruvate, several tca cycle and respiratory chain enzymes were significantly up-regulated. from these enzyme levels and the known regulatory network structure, we determined the probability for a given transcription factor to cause the coordinated expression changes. the most probable transcription factors to regulate the different degrees of respiration were gcr1p, cat8p, the rtg-proteins and the hap-complex. for the latter three ones we confirmed their importance for respiration by quantifying the degree of respiration and biomass yields in the corresponding deletion strains.  cat8p is required for wild-type like respiration, independent of its known activation of gluconeogenic genes. the rtg-proteins and the hap-complex are essential for wild-type like respiration under partially respiratory conditions. under fully respiratory conditions, the hap-complex, but not the rtg-proteins are essential for respiration. 