THE ROLE OF POLAR RESIDUES IN THE ADP/ATP CARRIER OF MITOCHONDRIA

Martin Klingenberg, Dörthe Heidkämper, Veronika Müller
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, 80336 Munich, Germany

The ADP/ATP carrier of mitochondria (AAC) has a high amount of negatively charged residues (bovine heart AAC, 40 positive, 21 negative). 70% are located towards the matrix site. Within the transmembrane helices are 4 positive but no negative residues. The construction of the AAC of three repeat domains entails a three repeat positioning of the majority of charged residues. Assuming pseudo circular arrangements of the three repeat domains charges form, the triads of charges may form charged rings. Strikingly is the charge ring of arginines within the second helix of each domain, further of acidic groups determinable of first helix in each domain, an x + x motif downstream a few residues from the first helix in each domain, which are largely obtained within the mitochondrial carrier family. These have been selected by mutagenesis by single residue charged neutralization. Mutagenesis led to serious damage of OxPhos in cells in mitochondria and to radical changes of translocational properties. Thus, the wt exchange mode pattern (ATP/ATP, ADP/ATP, etc.) is inverted by these mutations. The degree of mutational changes used is very strong within the triads causing a triad asymmetry. In each of the three charged rings one mutation affects only little the level of AAC expression. Each of these more resistant residues are located in different domains. The triad asymmetry rotates among the domains going from the cytosol to the matrix site and may cause a rotatory translocation along the alternating positive and negative charged rings. In spontaneous revertants (second site mutations) the second mutation neutralizes an opposing charge to the primary mutation which is located in an adjacent triad charged ring. These revertants in general enhance ATP versus ADP transport and revert exchange mode patterns. Since partners of the revertant charge pairs are located in different domains they may not directly interact but by network relayed charge propagation. In addition, the role of positive charges in binding cardiolipin and in steering the highly charged substrates into the carrier orifices will be discussed.