Two distinct membrane potential-dependent steps drive mitochondrial matrix protein translocation

Two driving forces energize precursor translocation across the inner mitochondrial membrane. Although the membrane potential (Ay) is considered to drive translocation of positively charged presequences through the TIM23 complex (presequence translocase), the activity of the Hsp70-powered import motor is crucial for the translocation of the mature protein portion into the matrix. In this study, we show that mitochondrial matrix proteins display surprisingly different dependencies on the Delta psi. However, a precursor's hypersensitivity to a reduction of the Delta psi is not linked to the respective presequence, but rather to the mature portion of the polypeptide chain. The presequence translocase constituent Pam17 is specifically recruited by the receptor Tim50 to promote the transport of hypersensitive precursors into the matrix. Our analyses show that two distinct Delta psi-driven translocation steps energize precursor passage across the inner mitochondrial membrane. The Delta psi- and Paml 17-dependent import step identified in this study is positioned between the two known energy-dependent steps: Delta psi-driven presequence translocation and adenosine triphosphate driven import motor activity.