Towards a molecular mechanism underlying mitochondrial protein import through the TOM-TIM23 complex
Mitochondria contain over a thousand different proteins, which, aside from a few encoded on the mitochondrial genome, are translated in the cytosol and targeted for import. For the majority, the first port of call is the translocase of the outer membrane (TOM-complex); their onward journey is via a procession of alternative molecular machines, conducting transport to their final sub-compartment destination: the outer-mitochondrial membrane (OMM), inner-mitochondrial membrane (IMM), inter-membrane space (IMS) or matrix. The pre-sequence translocase of the inner-membrane (TIM23-complex) is responsible for importing proteins with cleavable pre-sequences, and comes in two distinct forms: the TIM23SORT complex mediates IMM protein insertion and the TIM23MOTOR complex is responsible for matrix import. Progress in understanding these transport mechanisms has, until recently, 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 ∆ψ and ATP hydrolysis, the two main driving forces for transport 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 the PCP import time: passage of the 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 correlate poorly with the total amount of PCP imported - emphasising the importance of collecting rapid kinetic data to elucidating mechanistic detail. 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 health.