Protein phosphorylation in mitochondria - A study on fermentative and respiratory growth of Saccharomyces cerevisiae
ABSTRACT Phosphorylation as a posttranslational protein modification is a common subject of proteomic studies, but phosphorylation in mitochondria is still poorly investigated. The study presented here applied 2-DE to characterize phosphorylation in the yeast mitochondrial proteome and identified 59 spots corresponding to 34 phosphorylated mitochondrial or mitochondria-associated proteins. Most of these proteins presented putative substrates of mitogen-activated protein and target of rapamycin kinases, cAMP-dependent protein kinase, cyclin-dependent kinases and Snf1p suggesting them as key players in the phosphorylation of mitochondrial or mitochondria-associated proteins. The dynamic behaviour of the phosphoproteome under a major metabolic change, the shift from fermentation to respiration (diauxic shift), was further studied. Eight proteins (Ald4p, Eft1p/2p, Eno1p, Eno2p, Om14p, Pda1p, Qcr2p, Sdh1p) had growth dependent changes in their phosphorylation, indicating a role of phosphorylation-dependent regulation of translation, metabolic pathways (e.g. glucose fermentation, tricarboxylic acid cycle, pyruvate dehydrogenase and its bypass) and respiratory chain.
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ABSTRACT: The yeast Saccharomyces cerevisiae is a facultative aerobe able to adapt its metabolism according to the carbon substrate. The mechanisms of these adaptations involve at least partly the mitochondria but are not yet well understood. To address the possible role of protein phosphorylation event in their regulation, it is necessary in a first instance to determine precisely the phosphorylation sites that show changes depending on the carbon source. In this aim we performed an overall quantitative proteomic and phosphoproteomic study of isolated mitochondria extracted from yeast grown on fermentative (glucose or galactose) and respiratory (lactate) media. Label free quantitative analysis of protein accumulation revealed significant variation of 176 mitochondrial proteins including 108 proteins less accumulated in glucose medium than in lactate and galactose media. We also showed that the responses to galactose and glucose are not similar. Stable isotope dimethyl labeling allowed the quantitative comparison of phosphorylation levels between the different growth conditions. This study enlarges significantly the map of yeast mitochondrial phosphosites as 670 phosphorylation sites were identified, of which 214 were new and quantified. Above all, we showed that 90 phosphosites displayed a significant variation according to the medium and that variation of phosphorylation level is site-dependent. Biological Significance This proteomic and phosphoproteomic study is the first extensive study providing quantitative data on phosphosites responses to different carbon substrates independently of the variations of protein quantities in the yeast Saccharomyces cerevisiae mitochondria. The significant changes observed in the level of phosphorylation according to the carbon substrate open the way to the study of the regulation of mitochondrial proteins by phosphorylation in fermentative and respiratory media. In addition, the identification of a large number of new phosphorylation sites show that the characterization of mitochondrial phosphoproteome is not yet completed.Journal of proteomics 04/2014; 106. DOI:10.1016/j.jprot.2014.04.022 · 3.93 Impact Factor
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ABSTRACT: Protein phosphorylation is one of the major post-translational modifications to allow for signal transmission and fine tuning of metabolism on the cellular proteomic level. As such it is “one of the last instances” to modulate the activity of enzymes and hence to impact the cellular life irrespective of the basic conditions provided by the genome – and epigenome– controlled gene expression. The evolutionary increase in cellular complexity is reflected by highly sophisticated regulatory networks in multicellular eukaryotes based on the transfer of phosphate mostly onto the side chains of serine, threonine and tyrosine residues. Nature has chosen phosphate for inter- and intracellular communication, which is also an integral component of nucleic acids and can be regarded as the molecule of choice for life. Currently, life science is interested to unravel the network of reversible protein phosphorylation that is catalyzed by two antagonistic enzyme classes: the protein kinases and protein phosphatases. We are currently in the era of proteomics and enormously benefit from the progress of mass-spectrometry methods. This is documented by a huge number of “proteomic studies” that mostly provide a simple inventory of the existence of proteins – and/or their phosphorylated forms – under more or less defined conditions. So far, the physiological correlations could be established only in a few cases, e.g. by comparing two physiological conditions. Another strategy, which was addressed in this work, is the systematic screening of mutants defective in genes encoding either protein kinases or protein phosphatases. This approach benefits from the ease to predict these enzymes due to the presence of characteristic protein motifs. In combination with the major goal of this work – to shed light on the impact of protein phosphorylation in the mitochondrial (mt) compartment – the yeast Saccharomyces cerevisiae was chosen as a model system because of its respiro-fermentative metabolism, that allows for the maintenance of respiratory defective mutants. Indeed, this reverse genetic approach successfully revealed two kinases (Pkp1p, Pkp2p) and two phosphatases (Ppp1p, Ppp2p) as the key components regulating the pyruvate dehydrogenase complex by phosphorylation of serine 313 of its α- subunit Pda1p. In addition, evidence is provided that Pkp1p has an additional role in the assembly process of the PDH complex. Also, the effect of the deletion of the COQ8 gene (gene engaged in coenzyme Q synthesis; originally named ABC1) leading to respiratory deficiency, could be correlated with the phosphorylation of subunit Coq3p of the mitochondrial ubiquinone biosynthesis complex. 2 Finally, in the case of the kinase Sat4p (protein involved in salt tolerance), overexpression of the enzyme was used as an alternative approach to unravel the molecular basis of the originally observed salt sensitivity of sat4 mutants. The data suggest that Sat4p has a direct or indirect role in the late steps of iron-sulfur (Fe/S) cluster assembly of the so-called “aconitase-type” enzymes in mitochondria, accompanied by a strongly reduced steady state concentration of the Fe/S-cluster protein aconitase. Interestingly, a secondary phenotype became apparent upon overexpression of Sat4p: the abundance of the lipoic acid containing mitochondrial proteome was markedly reduced. Most likely this phenotype is due to the fact that the synthesis and/or attachment of lipoic acid depend on a Fe/S-cluster bearing enzyme. In the course of the work it became clear that the regulatory (mt) protein phosphorylation network of yeast evolved to meet the criteria of a life adapted to the ecological niche on temporarily available sugar rich sources. Clearly, the transfer of the respective data to higher eukaryotes is limited. However, it shows that yeast is primarily an excellent model system for the principal molecular reactions shared with higher eukaryotes.01/2013, Degree: postdoctoral lecture qualification (Habilitation), Supervisor: Gerhard Rödel
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ABSTRACT: The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt) localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE) approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p) and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p). The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p), Lys114 (Kgd2p) and Lys102 (Gcv3p), respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.PLoS ONE 08/2014; 9(8):e103956. DOI:10.1371/journal.pone.0103956 · 3.53 Impact Factor