Substrates for yeast mitochondrial cAMP-dependent protein kinase activity.

We showed that transcription of mitochondrial (mt) genes in Saccharomyces cerevisiae is governed in part by cellular cAMP levels, and that such transcriptional control is mediated via cAMP-dependent protein kinase (cAPK) activity. Here we use in vitro protein kinase assays with intact mitochondria from respiring cells to define protein substrates for mt cAPK. Our data show that there are at least eight mt proteins phosphorylated in a cAMP-dependent manner, ranging in M(r) from 96000 to 9500. Similar assays with organelles from an mtf1 mutant and its wild-type parent strain show no loss of any mt cAPK target proteins, suggesting that Mtflp (M(r) = 40000), the mt RNA polymerase specificity factor, does not require phosphorylation for activity. We further show, using double mutants for TPK1, TPK2, and TPK3, which encode catalytic subunits of the mt cAPK, that each of the eight mt substrate proteins is not phosphorylated equivalently by the individual catalytic subunits.