Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner-membrane (TIM23 complex) imports proteins with cleavable presequences, and comes in two flavours: the TIM23SORT complex mediates inner mitochondrial membrane (IMM) protein insertion and the TIM23MOTOR complex delivers proteins to the matrix. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time-resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how delta psi and ATP hydrolysis, the two main driving forces for import through the TIM23MOTOR complex, contribute to the import of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the OMM and initiation of IMM transport by the pre-sequence. The rates of these steps are influenced by PCP properties such as size and net charge, but not total amount of PCP imported; emphasising the importance of collecting rapid kinetic data, achieved here, to elucidate mechanistic detail. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel; presumably rapid success or failure of import is critical for maintaining mitochondrial fitness.
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