Cytochrome oxidase is a large multiprotein complex located in the inner mitochondrial membrane, where it functions as the terminal enzyme in the electron-transport chain (discussed in Chapter 14). (a) The first 18 amino acids of the precursor to subunit iV of this enzyme serve as a signal sequence for import of the subunit into the mitochondrion. (B) When the signal sequence is folded as an α helix, the positively charged amino acids (red) are clustered on one face of the helix, while the nonpolar ones (green) are clustered primarily on the opposite face. Uncharged polar amino acids are shaded orange; nitrogen atoms on the side chains of arg and Gln are colored blue. signal sequences that direct proteins into the matrix space always have the potential to form such an amphiphilic α helix, which is recognized by specific receptor proteins on the mitochondrial surface. (C) The structure of a signal sequence (of alcohol dehydrogenase, another mitochondrial matrix enzyme), bound to an import receptor (gray), as determined by nuclear magnetic resonance. The amphiphilic α helix binds with its hydrophobic face to  a hydrophobic groove in the receptor  (pDB code: 1om2).
660 Chapter 12: intracellular Compartments and protein sorting those inner membrane proteins that are synthesized within mitochondria. It also helps to insert some imported inner membrane proteins that are initially transported into the matrix space by the other complexes.
mitochondrial precursor proteins are imported as Unfolded polypeptide Chains
We have learned almost everything we know about the molecular mechanism of protein import into mitochondria from analyses of cell-free, reconstituted trans-location systems, in which purified mitochondria in a test tube import radiolabeled mitochondrial precursor proteins. By changing the conditions in the test tube, it is possible to establish the biochemical requirements for the import.
Mitochondrial precursor proteins do not fold into their native structures after they are synthesized; instead, they remain unfolded in the cytosol through interactions with other proteins. Some of these interacting proteins are general chaperone proteins of the hsp70 family (discussed in Chapter 6), whereas others are dedicated to mitochondrial precursor proteins and bind directly to their signal sequences. All the interacting proteins help to prevent the precursor proteins from aggregating or folding up spontaneously before they engage with the TOM complex in the outer mitochondrial membrane. As a first step in the import process, the import receptors of the TOM complex bind the signal sequence of the mitochondrial precursor protein. The interacting proteins are then stripped off, and the unfolded polypeptide chain is fed—signal sequence first—into the trans-location channel.
In principle, a protein could reach the mitochondrial matrix space by either crossing the two membranes all at once or crossing one at a time. One can distinguish between these possibilities by cooling a cell-free mitochondrial import system to arrest the proteins at an intermediate step in the translocation process. The result is that the arrested proteins no longer contain their N-terminal signal sequence, indicating that the N-terminus must be in the matrix space where the signal peptidase is located, but the bulk of the protein can still be attacked from outside the mitochondria by externally added proteolytic enzymes. Clearly, the precursor proteins can pass through both mitochondrial membranes at once to enter the matrix space (Figure 12–22). The TOM complex first transports the signal sequence across the outer membrane to the intermembrane space, where it binds to a TIM complex, opening the channel in the complex. The polypeptide chain is then either translocated into the matrix space or inserted into the inner membrane.