Endogenous protein phosphorylation in purified plant mitochondria

Purified mitochondria from potato (Solanum tuberosum L. cv Bintje) tubers were incubated with gamma-32P]ATP. Total 32P incorporation into proteins saturated after about 2 min and showed a Km (ATP) of 0.2 mM and a broad pH optimum of 6.5-8. About 30 polypeptides were labelled as shown by SDS-PAGE and autoradiography. The major labelled polypeptides were at 11, 14, 16 22-23, 40, 42 (the alpha-subunit of the pyruvate dehydrogenase complex), 45-46, 60, 62, 69, 84-86 and 97 kDa. By the use of atractylate, EGTA and trypsin the major phosphoproteins of 40 and 42 kDa and possibly some minor phosphoproteins in the range 26-33 kDa were localized to the matrix or the inner surface of the inner membrane. All other labelled polypeptides as well as (at least) two kinases (one Ca2(+)-dependent, the other Ca2(+)-independent) are outside the inner membrane.