Identification of the N-ethylmaleimide reactive protein of the mitochondrial phosphate transporter

Abstract

The mitochondrial phosphate carrier from Sarcophaga bullata thorax preparations is inhibited by the SH reagents p-(hydroxymercuri)benzoate [PHMB] and N-ethylmaleimide [NEM]. Based on an analysis utilizing dodecyl sulfate-polyacrylamide gels, an SH-containing 32,000 dalton protein was identified as a component of the phosphate carrier system. Two other 3H- NEM-labeled proteins of the inner mitochondrial membrane were eliminated from this role Wohlrab, and Greaney, on the basis that band IV (45,000 daltons) is absent from rat heart sonic submitochondrial particles and band VII (6500 daltons) does not react with PHMB. The mobility of the 32,000 dalton protein (0.43) is lower than that of the .gamma. subunit of the mitochondrial ATPase (0.46) and the carboxyatractyloside binding protein (0.48) on 12.5% dodecyl sulfate-polyacrylamide gels. In these flight muscle mitochondria, 0.87 nmol of 3H-NEM/nmol of cytochrome a is bound to the 32,000 dalton protein.