A Cooperative Action of the ATP-Dependent Import Motor Complex and the Inner Membrane Potential Drives Mitochondrial Preprotein Import

Abstract

The import of mitochondrial preproteins requires an electric potential across the inner membrane and the hydrolysis of ATP in the matrix. We assessed the contributions of the two energy sources to the translocation driving force responsible for movement of the polypeptide chain through the translocation channel and the unfolding of preprotein domains. The import-driving activity was directly analyzed by the determination of the protease resistances of saturating amounts of membrane-spanning translocation intermediates. The ability to generate a strong translocation-driving force was solely dependent on the activity of the ATP-dependent import motor complex in the matrix. For a sustained import-driving activity on the preprotein in transit, an unstructured N-terminal segment of more than 70 to 80 amino acid residues was required. The electric potential of the inner membrane was required to maintain the import-driving activity at a high level. The electrophoretic force of the potential exhibited only a limited capacity to unfold preprotein domains. We conclude that the membrane potential increases the probability of a dynamic interaction of the preprotein with the import motor. Polypeptide translocation and unfolding are mainly driven by the inward-directed translocation activity based on the functional cooperation of the import motor components.Although mitochondria can synthesize a small subset of proteins on their own, the vast majority have to be imported after their synthesis in the cytosol (11,23,36,40). The precursor proteins are transported through specific translocase complexes residing in the outer and inner membranes (21, 37). However, the relatively small inner diameter of the translocation channels presents a major constraint for the import process (17, 49). Experimental evidence indicates that preproteins destined for the mitochondrial matrix cross the membranes in an extended conformation (42). Posttranslational import of mitochondrial preproteins therefore requires the unfolding of folded preprotein domains prior to or during the translocation reaction.Two mitochondrial energy sources are responsible for both vectorial movement of the precursor polypeptide in the translocation channel and unfolding of preproteins. The insertion of the amino-terminal segment of the preprotein into the inner membrane is dependent on the electric potential (⌬) across the inner membrane. It is thought that the potential exerts an electrophoretic force on the positively charged N-terminal targeting sequence (29). In addition, the ⌬ has been shown to be involved in the function of the TIM23 protein complex (2). The full translocation of the polypeptide chain requires a second energy source, the hydrolysis of matrix ATP. The enzymatic machinery that couples preprotein translocation and ATP hydrolysis is provided by the import motor complex, which is located at the inner face of the inner membrane in the direct vicinity of the TIM23 translocation channel (36,39,48,55). Its core component is the main mitochondrial chaperone...

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

“…The requirement for mtHsp70 in granzyme mitochondrial import is less surprising as GA and GB have been shown to interact strongly with cytosolic Hsp27, Hsp70 and Hsp90. [69][70][71] SSC1-3 allele presents a G56S mutation in the mtHsp70 ATPase domain, 72 indicating that GB might cross the inner mitochondrial membrane in a forceful manner. Moreover SSC1-2 yeast strain carries an allele of mtHsp70 that poorly interact with Tim44 at 37°C.…”

A systematic review of evidence for silver nanoparticle-induced mitochondrial toxicity

“…Sufficient mitochondrial membrane polarization is required for essential mitochondrial functions, including canonical protein import mechanisms, 65,66 ATP production, 67 and proper responses to cellular calcium concentration flux. 68 Loss of Δψm is the most frequently studied mitochondrial outcome as a result of AgNP exposure.…”

Impaired Interdomain Communication in Mitochondrial Hsp70 Results in the Loss of Inward-directed Translocation Force

“…Apart from driving polypeptide movement through the translocation channel, mtHsp70 is also required for the unfolding of stably folded preprotein domains (26). Active unfolding implies the generation of an inward-directed translocation force on preproteins in transit (27,29). As was previously observed for the conditional mutant ssc1-2, in the absence of a translocation force import becomes limited by (low) spontaneous unfolding rates.…”

“…3C). To maximally challenge the import machinery, saturating amounts of recombinant precursor proteins purified from E. coli cells were utilized (29). We employed mitochondria isolated from ssc1-3 cells expressing either wild-type or the NBD mutants as described above.…”

“…For the analysis of the inward-directed import activity, recombinant precursor proteins (b 2 (167) ⌬ -DHFR) were imported in saturating amounts (29) in the presence of the dihydrofolate reductase (DHFR) ligand methotrexate (MTX) as described (45). After terminating the import reaction by addition of valinomycin, mitochondria were re-isolated and resuspended in import buffer in the presence of ATP.…”