As noted previously and as indicated in Figure 1, ATP hydrolysis by Hsp70 chaperone proteins in both the cytosol and the mitochondrial matrix is required for the import of mitochondrial proteins. Cytosolic Hsp70 expends energy to maintain bound precursor proteins in an unfolded state so that they can be translocated into the matrix. The importance of ATP to this function was demonstrated in studies in which a mitochondrial precursor protein was purified and then denatured (unfolded) by urea. When tested in the cell-free mitochondrial translocation system, the denatured protein was incorporated into the matrix in the absence of ATP. In contrast, the same precursor protein in its native, undenatured state was not imported in the absence of ATP, even in the presence of cytosolic chaperones.
Fig1. Protein import into the mitochondrial matrix. Precursor proteins synthesized on cytosolic ribosomes are maintained in an unfolded or partially folded state by bound chaperones, such as cytosolic Hsp70 (step 1). After a precursor protein binds to an import receptor near a site of contact with the inner membrane (step 2), it is transferred into the general import pore (step 3). The translocating protein then moves through this channel and an adjacent channel in the inner membrane (steps 4–5). Note that translocation occurs at rare “contact sites” at which the inner and outer membranes appear to touch. Binding of the translocating protein by matrix Hsp70 and subsequent ATP hydrolysis by Hsp70 helps drive import into the matrix. Once the targeting sequence is removed by a matrix protease and Hsp70 is released from the newly imported protein (step 6), the protein folds into its mature, active conformation within the matrix (step 7). Folding of some proteins depends on matrix chaperonins. See G. Schatz, 1996, J. Biol. Chem. 271:31763, and N. Pfanner et al., 1997, Annu. Rev. Cell Dev. Biol. 13:25.
The sequential binding to and ATP-driven release of multiple matrix Hsp70 molecules from a translocating protein may simply trap the unfolded protein in the matrix. Alter natively, the matrix Hsp70, anchored to the membrane by the Tim44 protein, may act as a molecular motor to pull the protein into the matrix (see Figure 1). In this case, the functions of matrix Hsp70 and Tim44 would be analogous to those of the chaperone BiP and Sec63 complex, respectively, in post-translational translocation into the ER lumen.
The third energy input required for mitochondrial protein import is a H+ electrochemical gradient, or proton- motive force, across the inner membrane. Recall from Chapter 12 that protons are pumped from the matrix into the intermembrane space during electron transport, creating an electric potential across the inner membrane. In general, only mitochondria that are actively undergoing respiration, and therefore have generated a proton-motive force across the inner membrane, are able to translocate precursor proteins from the cytosol into the mitochondrial matrix. Treatment of mitochondria with inhibitors or uncouplers of oxidative phosphorylation, such as cyanide or dinitro phenol, dissipates this proton-motive force. Although pre cursor proteins can still bind tightly to receptors on such poisoned mitochondria, the proteins cannot be imported, either in intact cells or in cell-free systems, even in the presence of ATP and chaperone proteins. Scientists do not fully understand how the proton-motive force is used to facilitate entry of a precursor protein into the matrix. Once a protein is partially inserted into the inner membrane, it is subjected to a membrane potential of 200 mV (matrix negative). This seemingly small potential is established across the very narrow hydrophobic core of the lipid bilayer, which gives an enormous electrochemical gradient, equivalent to about 400,000 V/cm. One hypothesis is that the positive charges in the amphipathic matrix-targeting sequence are simply “electrophoresed,” or pulled, into the matrix by the inside-negative membrane potential.
