The Mitochondrial Carrier Family (MCF) groups integral membrane proteins that transport various metabolites across the inner mitochondrial membrane. This transport is needed in major metabolic pathways occurring in mitochondria and is essential in eukaryotic metabolism. The mis-function of several identified human mitochondrial carriers has been associated with severe diseases. In order to achieve a better understanding of mitochondrial carriers two different families of mitochondrial carriers where chosen to be studied: the AACs (ADP/ATP Carriers) and the UCPs (UnCoupling Proteins). Two heterologous expression systems of these carriers were developed: the cell free and the expression in E. coli as fusion proteins. The cell free system allowed the production and purification of about 0,6 mg of protein per milliliter of reaction mixture and the expression in E.coli proved to be very efficient for the functional characterization of ADP/ATP carriers. A functional test was developed for the uncoupling protein also. This test, based on the measure of electrical currents generated by the transport activity of UCP, allowed the functional characterization of native UCP1.
