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Abstract

Mitochondria import the vast majority of proteins from the cytosol. Protein translocation machineries in outer and inner membranes facilitate precursor recognition and transport. Most mitochondrial proteins utilize N-terminal presequences as targeting signals that eventually direct them across the inner mitochondrial membrane. These precursors are transported by the TOM complex across the outer-, and subsequently by the TIM23 complex across the inner membrane. During this process the translocases align and the polypeptide chain is translocated across both membranes in a coupled manner. A transient precursor-containing TOM-TIM23 supercomplex is formed. This TOM-TIM23 supercomplex provides a fascinating import intermediate which can be stabilized if the precursor contains a tightly folded moiety at the C-terminus that is not able to pass through the TOM complex. Such a supercomplex can be generated during in vitro import, and in vivo. The stabilized TOM-TIM23 supercomplex can be purified for downstream analysis. The possibility of pausing translocation at this step provides a means to understand the mechanisms underlying precursor translocation.