Protein phosphorylation in plant mitochondria

Abstract: Protein phosphorylation in the subcompartments of plant mitochondria was investigated by labelling with [gamma-32P]ATP and by SDS-PAGE/autoradiography. About 20 proteins in inside-out inner mitochondrial membranes from potato tubers were phosphorylated by endogenous protein kinases when incubated with [gamma-32P]ATP. None of the phosphorylated proteins were labelled when using [alpha-32P]ATP as a phosphate donor. The reversible protein phosphorylation in these membranes is mainly a result of the action of inner membrane serine/threonine protein kinases/phosphatases, although the dephosphorylation of inner membrane proteins appears to be catalysed by protein phosphatases located in the matrix. In addition, a 37 kDa protein contained acid-labile phosphate groups, indicating modification of histidine residues. The phosphorylation of inner membrane proteins requires divalent cations (i.e. Mg2+ or Mn2+) and is stimulated under oxidising conditions. Two autophosphorylated inner membrane proteins with molecular mass of 16.5 and 30 kDa are putative protein kinases. Two inner membrane phosphoproteins with molecular mass of 22 and 28 kDa were identified by N-terminal sequencing as the delta?-subunit and b-subunit, respectively, of the FoF1-ATPase. A 17 kDa phosphoprotein has some characteristics of a nucleoside diphosphate kinase. All other plant inner mitochondrial membrane phosphoproteins remain to be identified. A 39.5 and 41 kDa protein are the most heavily labelled phosphoproteins in the matrix of potato tuber mitochondria. The 41 kDa protein is probably the alpha-subunit of pyruvate dehydrogenase, and the 39.5 kDa protein, which was also labelled with 32Pi, may be the alpha-subunit of succinyl-CoA synthetase. In pea leaf mitochondria, a 17.4 kDa protein, which is probably located in the intermembrane space, was purified and identified as a nucleoside diphosphate kinase. This pea protein is autophosphorylated on both the catalytic histidine and on serine residues. The labelling on serine residues was higher then for the histidine, and kinetic studies demonstrated a faster rate of phosphorylation of serine compared to histidine.