Phosphorylation-dephosphorylation of pyruvate dehydrogenase from bakers' yeast

Abstract

The pyruvate dehydrogenase complex was purified to homogeneity from bakers' yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase kinase activity was detected at any stage of the purification. However, the purified pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. The protein-bound radioactivity was localized in the pyruvate dehydrogenase alpha subunit. The phosphorylated, inactive pyruvate dehydrogenase complex was dephosphorylated and reactivated with purified pyruvate dehydrogenase phosphatase from bovine heart. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide, which was purified to homogeneity. The sequence of the phosphopeptide was established to be Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphotetradecapeptide derived from the alpha subunit of bovine kidney and heart pyruvate dehydrogenase: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

Regulation of the PDA1 gene encoding the E1α subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae

“…However, since overproduction of the Ela subunit increases the specific activity of the complex, post-translational modification of the Ela subunit seems the more likely possibility. This posttranslational modification might be similar to that in higher eukaryotes (Yeaman et al, 1978;Sugden et al, 1979) as has been proposed previously (Uhlinger et al, 1986), i.e. phosphorylation of specific serine residues in the Ela subunit.…”

“…Indeed, in higher eukaryotes, the activity of the PDH complex is mainly regulated via phosphorylatioddephosphorylation of specific serine residues in the E l a subunit (Yeaman et al, 1978;Sugden et al, 1979). The E l a subunit of the S. cerevisiae PDH complex also contains a specific serine residue which can be phosphorylated in vitro, using a heterologous kinase (Uhlinger et al, 1986); however, such regulation in vivo has not yet been shown.…”

The pyruvate dehydrogenase complex of Saccharomyces cerevisiae is regulated by phosphorylation

“…Previous attempts to detect yeast PDC kinase [7,8] have been unsuccessful, probably because PDC has been purified from bakers' yeast strains, as it now appears that yeast must be cultured under carefully defined conditions to detect phosphorylation of the complex. Moreover, in our hands, it has proved difficult to assay PDC kinase routinely in crude mitochondrial extracts using endogenous yeast PDC, purified exogenous yeast or bovine PDC as substrates with reproducible phosphorylation only occurring in intact mitochondria.…”

Profiling Phosphoproteins of Yeast Mitochondria Reveals a Role of Phosphorylation in Assembly of the ATP Synthase

“…It is evident that these processes have to be tightly regulated to permit a mitochondrial response to changes in energy demand, cellular metabolism, or environmental conditions (6, 7). Until recently the most common regulatory mechanism of eukaryotic cells, reversible phosphorylation (8 -10), was considered to represent an exception in the case of mitochondria, including the E1 subunit of pyruvate dehydrogenase and the branched-chain ␣-ketoacid dehydrogenase (11)(12)(13)(14).A number of recent studies have provided evidence that phosphorylation of mitochondrial proteins is much more frequent than expected (15-23). (i) Incubation of isolated mitochondria with radiolabeled ATP or staining of mitochondrial proteins with phosphospecific dyes suggested that a substantial fraction of mitochondrial proteins are phosphorylated (24 -27).…”