Energy to Drive Translocation

Mitochondrial protein translocation is fueled by ATP hydrolysis and electrochemical potential across the inner membrane. The electrochemical potential of 200 millivolts pulls positively charged presequences through the TIM channel, while ATP hydrolysis by mitochondrial Hsp70 provides the mechanical force needed to translocate the polypeptide chain into the matrix.

In the thermal ratchet model, an emerging polypeptide chain moves back and forth across the TIM channel. When ATP bound to Hsp70 is hydrolyzed, it prevents further polypeptide backsliding. As multiple Hsp70s bind sequentially, the precursor is translocated forward into the mitochondrial matrix.

In the cross-bridge ratchet model, matrix Hsp70 binds the TIM complex near its channel mouth. ATP binding triggers a conformational change that latches Hsp70 onto the emerging precursor. ATP hydrolysis secures this binding and pulls the peptide through the TIM channel, while ATP rebinding causes Hsp70 dissociation and peptide release into the matrix.

Precursor proteins undergo unfolding through two distinct mechanisms. Precursors with shorter presequences undergo spontaneous global unfolding. In contrast, precursors with longer positively charged presequences undergo local unfolding, allowing the unstructured presequence to traverse the TOM/TIM import complexes and interact with negative charges on the inner membrane.

mtHsp70 traps loosely folded precursors without undergoing conformational change, translocating them directly to the matrix. In contrast, tightly folded polypeptides induce ATP-dependent conformational changes in mtHsp70. mtHsp70 uses ATP hydrolysis energy to pull tightly folded peptides across the TIM translocase, with ATP rebinding causing mtHsp70 opening and precursor release.

Mitochondrial Hsp70 associates with TIM44 to recognize the emerging polypeptide at the TIM channel. This mtHsp70-TIM44 complex accelerates precursor unfolding and translocates the polypeptide entirely into the matrix. The interaction facilitates efficient coupling of ATP hydrolysis to mechanical pulling force during translocation.

Accelerated precursor unfolding, coupled to ATP-dependent conformational changes in mtHsp70, generates pulling force that drives unidirectional forward movement. This mechanism prevents backward movement and ensures efficient transport into the matrix. The combination of unfolding and ATP-dependent pulling creates a ratchet-like mechanism that maintains directionality throughout translocation.