Biogenesis of the mitochondrial ABC-transporter Mdl1 involves mitochondrial import and export machineries

Mitochondrial preproteins with N-terminal signal sequences are imported across or into the mitochondrial inner membrane by the presequence translocase. The membrane-embedded core of the presequence translocase (Tim23, Tim17, and Tim50) mediates lateral membrane insertion of simple single-spanning membrane proteins by a stop-transfer mechanism. In contrast, complex polytopic membrane proteins are believed to be inserted into the membrane by the so-called conservative sorting pathway. This model proposes initial full translocation across the membrane by the presequence translocase and the associated import motor PAM. In a subsequent step, the precursor is proposed to insert into the membrane from the matrix side with the help of the protein export translocase Oxa1. We have investigated biogenesis of the S. cerevisiae ABC-transporter Mdl1, a protein with six a-helical transmembrane segments and a large C-terminal matrix domain. We found that Mdl1 requires both the lateral sorting as well as the conservative sorting mechanism, with distinct modules of the Mdl1 transmembrane domain following different pathways. We conclude that acquisition of the final transmembrane topology of Mdl1 is a multi-step process that involves the sequential action of different protein import and export machineries.