The Protein Component(s) of the Isolated Phosphate-Transport System of Mitochondria

Abstract

The ethylmaleimide-sensitive phosphate-transport system of [rat] heart mitochondria was isolated and the activity reconstituted in liposomes. The identification of the phosphate-carrier protein was complicated by several factors. The phosphate-carrier fraction, isolated through different procedures in different laboratories, contains 4-5 protein components of very similar apparent MW, as shown by gradient dodecylsulfate gel electrophoresis. The amino acid composition of protein 1, 2 and 3 (apparent MW = 34,500, 34,000 and 33,000, respectively) is similar although not identical. Including several protease inhibitors during isolation of the phosphate-carrier fraction does not influence the protein pattern. Labeled N-ethylmaleimide binds only protein 1 and 3, labeled N,N''-dicyclohexylcarbodiimide bonds only to protein 1 and 2. N,N''-Dicyclohexylcarbodiimide had no effect on the reconstituted phosphate-exchange activity after incubation with intact mitochondria or proteoliposomes. In the presence of protecting mersalyl concentrations N-ethylmaleimide inhibits the phosphate transport in intact mitochondria. Under these conditions no label ethylmaleimide was bound to the phosphate-carrier fraction. Comparison of the reconstituted transport activity with the protein pattern of fractions, isolated with or without cardiolipin, shows correlation of transport with the amount of protein 2 but not with that of protein 1, which binds ethylmaleimide. A model of the mitochondrial phosphate carrier is presented which suggests a proteolytic degradation of the phosphate-carrier protein during its isolation. The model explains the contradictory results of this investigation.