INSERTION INTO THE MITOCHONDRIAL INNER MEMBRANE OF A POLYTOPIC PROTEIN, THE NUCLEAR-ENCODED OXA1P

Oxa1p, a nuclear-encoded protein of the mitochondrial inner membrane w ith five predicted transmembrane (TM) segments is synthesized as a pre cursor (pOxa1p) with an N-terminal presequence, It becomes imported in a process requiring the membrane potential, matrix ATP, mt-Hsp70 and the mitochondrial processing peptidase (MPP). After processing, the ne gatively charged N-terminus of Oxa1p (similar to 90 amino acid residue s) is translocated back across the inner membrane into the intermembra ne space and thereby attains its native N-out-C-in orientation, This e xport event is dependent on the membrane potential. Chimeric preprotei ns containing N-terminal stretches of increasing lengths of Oxa1p fuse d on mouse dehydrofolate reductase (DHFR) were imported into isolated mitochondria. In each case, their DHFR moieties crossed the inner memb rane into the matrix. Thus Oxa1p apparently does not contain a stop tr ansfer signal. Instead the TM segments are inserted into the membrane from the matrix side in a pairwise fashion, The sorting pathway of pOx a1p is suggested to combine the pathways of general import into the ma trix with a bacterial-type export process, We postulate that at least two different sorting pathways exist in mitochondria for polytopic inn er membrane proteins, the evolutionarily novel pathway for members of the ADP/ATP carrier family and a conserved Oxa1p-type pathway.