THE PHOSPHATE CARRIER FROM YEAST MITOCHONDRIA - DIMERIZATION IS A PREREQUISITE FOR FUNCTION

Wild type phosphate carrier (PIC) from Saccharomyces cerevisiae and re combinant PIC proteins with different C-terminal extensions were expre ssed in Escherichia coli as inclusion bodies. From these, PIC was isol ated with the detergent sodium lauroyl sarcosinate in a form, partiall y monomeric and unfolded. This PIC associates to stable dimers after e xchanging the detergent to the polyoxyethylene detergent C12E8 and dia lysis. Combining two differently tagged monomers of PIC and following this with affinity chromatography yields defined homo-and heterodimeri c forms of PIG, which are all fully active after reconstitution. As a member of the mitochondrial carrier family PIC is supposed to function as a homodimer, We investigated its dimeric nature in the functionall y active state after reconstitution. When reconstituting PIC monomers a sigmoidal dependence of transport activity on the amount of inserted protein is observed, whereas insertion of PIC dimers leads to a linea r dependence. Heterodimeric PIC constructs consisting of both an activ e and an inactivated subunit do not catalyze phosphate transport. In c ontrast, reconstitution of a mixture of active and inactive monomeric subunits led to partially active carrier. These experiments prove (i) that PIC does not function in monomeric form, (ii) that PIC dimers are stable both in the solubilized state and after membrane insertion, an d (iii) that transport catalyzed by PIC dimers involves functional cro ss-talk between the two monomers.