In another transport route to the inner membrane or intermembrane space, the TIM23 complex initially translocates the entire protein into the matrix space (Figure 12–25B). A matrix signal peptidase then removes the N-terminal signal sequence, exposing a hydrophobic sequence at the new N-terminus. This signal sequence guides the protein to the OXA complex, which inserts the protein into the inner membrane. As mentioned earlier, the OXA complex is primarily used to insert proteins that are encoded and translated in the mitochondrion into the inner membrane, and only a few imported proteins use this pathway. Translocators that are closely related to the OXA complex are found in the plasma membrane of bacteria and in the thylakoid membrane of chloroplasts, where they insert membrane proteins by a similar mechanism.
Many proteins that use these pathways to the inner membrane remain anchored there through their hydrophobic signal sequence (see Figure 12–25A,B). Others, however, are released into the intermembrane space by a protease that removes the membrane anchor (Figure 12–25C). Many of these cleaved proteins remain attached to the outer surface of the inner membrane as peripheral subunits of protein complexes that also contain transmembrane proteins.
Certain intermembrane-space proteins that contain cysteine motifs are imported by a yet different route. These proteins form a transient covalent disulfide bond to the Mia40 protein (Figure 12–25D). The imported proteins are then released in an oxidized form containing intrachain disulfide bonds. Mia40 becomes reduced in the process, and is then reoxidized by passing electrons to the electron transport chain in the inner mitochondrial membrane. In this way, the energy stored in the redox potential in the mitochondrial electron transport chain is tapped to drive protein import.
Mitochondria are the principal sites of ATP synthesis in the cell, but they also contain many metabolic enzymes, such as those of the citric acid cycle. Thus, in addition to proteins, mitochondria must also transport small metabolites across their membranes. While the outer membrane contains porins, which make the membrane freely permeable to such small molecules, the inner membrane does not. Instead, a family of metabolite-specific transporters transfers a vast number of small molecules across the inner membrane. In yeast cells, these transporters comprise a family of 35 different proteins, the most abundant of which transport ATP, ADP, and phosphate. These are multipass transmembrane proteins, which do not have cleavable signal sequences at their N-termini but instead contain internal signal sequences. They cross the TOM complex in the outer membrane, and intermembrane-space chaperones guide them to the TIM22 complex, which inserts them into the inner membrane by a process that requires the membrane potential, but not mitochondrial hsp70 or ATP (Figure 12–25E). An energetically favorable partitioning of the hydrophobic transmembrane regions into the inner membrane is likely to drive this process.
Two signal sequences Direct proteins to the Thylakoid membrane in Chloroplasts
Protein transport into chloroplasts resembles transport into mitochondria. Both processes occur post-translationally, use separate translocation complexes in each membrane, require energy, and use amphiphilic N-terminal signal sequences that are removed after use. With the exception of some of the chaperone molecules, however, the protein components that form the translocation complexes differ. Moreover, whereas mitochondria harness the electrochemical H+ gradient across their inner membrane to drive transport, chloroplasts, which have an electrochemical H+ gradient across their thylakoid membrane but not their inner membrane, use GTP and ATP hydrolysis to power import across their double membrane. The functional similarities may thus result from convergent evolution, reflecting the common requirements for translocation across a double membrane.