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Nuclear encoded mitochondrial precursors are imported to the inner membrane in a multistep process involving two separate translocons, TIM22 and TIM23. TIM23 is a cation-selective pore that remains closed by the N terminal segment of the protein. Negative charges on the TIM23 act as a receptor for the incoming precursor, pulling the positively charged matrix-targeting sequence for peptide insertion and translocation.

Transport of mitochondrial precursors across the TIM23 channel is driven by the energy of ATP hydrolysis. As TIM23 associates with TIM44 towards the matrix side of the inner membrane, the TIM44 dimer recruits two molecules of matrix Hsp70 that bind to the mouth of the TIM23 channel. Like a motor, Hsp70 pulls the emerging peptide out of the TIM23 channel, releasing it into the matrix.

TIM22 is also a voltage-activated channel but recognizes carrier proteins and integral membrane proteins without a matrix targeting sequence. Carrier proteins have a non-cleavable presequence and multiple internal import signals. They contain a specific structural motif called the carrier signature adjacent to the transmembrane segments of the proteins. During precursor import, TOM70 recognizes the internal signals while TIM10 and TIM12 recognize carrier signatures. However, unlike TIM23, translocation across TIM22 is driven by the electrochemical potential across the inner membrane unaided by chaperones or ATP hydrolysis energy.

A third translocase, the oxidase assembly (OXA), is required for inserting integral membrane proteins translocated by the TIM22/23 channels. Single spanning inner membrane proteins with cleavable N-terminal presequence follow the stop-transfer route and are integrated into the inner membrane by the lateral release of the blocked precursor from the TIM23 channel. Oxa substrates include multispanning inner membrane proteins with cleavable presequence and several carrier proteins with non-cleavable internal import signals. Precursors that are translocated into the matrix are reinserted into the inner membrane through the Oxa translocase following the conservative route of mitochondrial protein sorting.

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