A study is presented on cyclic adenosine monophosphate- (cAMP-) dependent phosphorylation of mammalian mitochondrial proteins. Immunodetection with specific antibodies reveals the presence of the catalytic and the regulatory subunits of cAMP-dependent protein kinase (PKA) in the inner membrane and matrix of bovine heart mitochondria. The mitochondrial cAMP-dependent protein kinase phosphorylates mitochondrial proteins of 29, 18, and 6.5 kDa. With added histone as substrate, PKA exhibits affinities for ATP and cAMP and pH optimum comparable to those of the cytosolic PKA. Among the mitochondrial proteins phosphorylated by PKA, one is the nuclear-encoded (NDUFS4 gene) 18 kDa subunit of complex I, which has phosphorylation consensus sites in the C terminus and in the presequence. cAMP promotes phosphorylation of the 18 kDa subunit of complex I in myoblasts in culture and in their isolated mitoplast fraction. In both cases cAMP-dependent phosphorylation of the 18 kDa subunit of complex I is accompanied by enhancement of the activity of the complex. These results, and the finding of mutations in the NDUFS4 gene in patients with complex I deficiency, provide evidence showing that cAMP-dependent phosphorylation of the 18 kDa subunit of complex I plays a major role in the control of the mitochondrial respiratory activity.

In bovine heart mitochondria a protein of M, 18 kDa, phosphorylated by mtPKA, is associated to the NADH-ubiquinone oxidoreductase in the inner membrane and is present in purified preparation of this complex. The 18 kDa phosphoprotein has now been isolated and sequenced. It is identified as the 18 kDa (IP) AQDQ subnnit of complex I, a protein of 133 amino acids with a phospborylation consensus site RVS at position 129-131.

In intact bovine heart mitochondria, cAMP-dependent phosphorylation of 42, 29, 18 and 6.5 kDa proteins was inhibited by carboxyatractyloside. This shows that both mitochondriai cAMP-dependent protein kinase (mtPKA) and its protein substrates are localized at the matrix side of the inner mitochondrial membrane. Proteins of 42, 29, 18, and 6.5 kDa were also bound at the outer surface of mitochondria where they were phosphorylated by the added purified catalytic subunit of PKA. In the cytosol from bovine heart proteins of the above molecular weights were phosphorylated by the cytosolic PKA.