(43) THE MITOCHONDRIAL PHOSPHATE CARRIER: STUDYING THE SUBSTRATE PATHWAY BY THE USE OF DEFINED HETERODIMERS

R. Kraemer, A. Schroers and H. Wohlrab
Institute of Biochemistry, University of Cologne, 50674 Koeln, Germany and Boston Biomedical Research Institute, Boston, MS 02114, USA

We have heterologously expressed wild type and mutant phosphate carrier (PIC) from yeast mitochondria in E. coli inclusion bodies. PIC-constructs with different molecular tags were solubilized from inclusion bodies, purified and functionally reconstituted into proteoliposomes. Furthermore, we have developed a method to isolate the tagged proteins as monomers, to construct defined dimers (homodimers and heterodimers), and to isolate specific dimeric forms from mixtures of different constructs. Titration of proteoliposomes with PIC and functional analysis of reconstituted heterodimers led to the following conclusions. (i) Dimerization is an essential prerequisite for function of PIC. (ii) The dimeric form of the carrier is stable, no dynamic equilibrium with monomers is observed. (iii) A functional crosstalk between the individual monomers is observed. Heterodimers were used to investigate the catalytic function of PIC. We have previously shown that a reversible functional shift between coupled and uncoupled transport mode (efflux) depends on modification of a single cysteine at position 28 in the first transmembrane segment. We replaced this cysteine with various other amino acids both in the homodimeric (both monomers mutated) and heterodimeric form (only one monomer mutated). In addition, amino acids in the neighborhood of cys28 were replaced (scanning mutagenesis). The following conclusions can be drawn. (i) A positive charge in the middle part of the first transmembrane segment converts PIC from coupled transport to the efflux mode. (ii) Replacement of one single amino acid in position 28 (heterodimer) is sufficient for the functional shift. (iii) Amino acids at particular positions of the first transmembrane segment show close functional interaction (monomer-monomer). These results will be discussed in terms of topology and functional consequences.