Cooperation of stop-transfer and conservative sorting mechanisms in mitochondrial protein transport

The mitochondrial inner membrane is a highly protein-rich membrane with central importance for oxidative phosphorylation and metabolite transport. A large number of inner-membrane proteins are synthesized as preproteins with cleavable presequences. Opposing mechanisms of preprotein insertion into the membrane have been debated: stop-transfer with arrest in the inner membrane versus conservative sorting via the matrix. We dissected the membrane insertion of a multispanning ABC transporter. The N-terminal membrane domain was laterally released from the presequence translocase of the inner membrane (TIM23 complex) by a stop-transfer mechanism, whereas the subsequent domain was imported via the matrix heat-shock protein 70 (mtHsp70) motor and exported by the oxidase assembly (OXA) translocase. These observations lead to an unexpected solution to the controversial debate about mitochondrial preprotein sorting. Stop-transfer and conservative sorting are not mutually exclusive pathways but represent sorting mechanisms that cooperate in the membrane integration of a protein with complex topology. We conclude that the multispanning protein is inserted in a modular manner by the coordinated action of two inner-membrane preprotein translocases.