If the inner membrane is so impermeable, how do proteins enter?

The outer membrane of the mitochondria contains the protein "porin". This forms an aqueous channel through which proteins up to 10,000 daltons can pass and go into the intermembrane space. Most proteins cannot get into the matrix unless they pass through the inner membrane. This membrane contains cardiolipin which renders it virtually impermeable. This requires transport mechanisms across the membrane that are more organized and regulated. A very simple view of the process is diagrammed in this cartoon. This figure is taken from Alberts et al, Molecular Biology of the Cell, Garland Publishing, N.Y. 1994, Third Edition
Mitochondrial import signals.

Most proteins must be uncoiled or stretched out to go through the translocators.  This involves ATP binding and is monitored and stabilized by a chaperone proteins, including hsp70.  Thus, before the protein can go through Tom complex, it must become "translocation competent".  

Transport through the outer membrane: characteristics of Tom complex.  
Not surprisingly, the TOM complex will include import receptors that initially recognize the signal peptide or a signal sequence (these include Tom20, Tom22, and Tom70). Different proteins use different receptors. In the above cartoon, the receptor is represented as a blue oval in which the signal peptide is inserted. The receptors then bring the protein to the region containing the translocator proteins. This is actually a complex of proteins.  

It is called the General Import Pore (GIP) and it facilitates the translocation of the presequence of the protein across the outer membrane. (the GIP is made of Tom40, Tom5, Tom 6, and Tom7). Tom40 appears to be the core element of the pore.  It also interacts with polypeptide chains passing through the pore.  All of the other Tom components in GIP are anchored to the outer membrane by helical transmembrane segments .  

Characteristics of Tim Complexes
Mitochondrial proteins destined for the matrix often have a cleavable signal peptide on the protein which must be recognized before it will be admitted through the mitochondrial translocator. These proteins with "amino terminal signals" , or "preproteins"  or "presequences" (current literature) usually interact with Tom20 first. Then, they have to get through the outer membrane.  To do that, they are transferred to the GIP complex: First, they interact with Tom22 and Tom5 which ushers them to the pore formed by Tom40. They then enter the matrix  using the pore complex.

Entry of proteins is dependent on the hydrogen pumps (electron transport chain)
 Also, very important, their entry is dependent on membrane potential.  This is set up by the electron transport complexes.  Recall that hydrogen ions are being pumped into the intermembrane space creating a charge gradient that is more negative on the matrix site.  This membrane potential actually helps pulls the protein into the GIP.

The protein then enters the matrix where the cleavable preprotein is clipped off by a protease and a chaperone protein ( mt-hsp70) in the matrix works with Tim44 to complete the full transfer to the matrix. This chaperone mthsp70 and Tim 44 actually "pull" the protein into the matrix by a process that requires ATP.  It also requires the membrane potential set up by the electron transport chain. Negative charges in the matrix, set up by the pumping of hydrogen ions to the inter cristal space, attract the protein which has positive charges on the end that enters the GIP.

Some mitochondrial proteins destined for the inner membrane have a cleavable signal peptide followed by one or more  membrane-spanning segments that  serve to insert the polypeptide into the inner membrane after it gets in the matrix. This is how the electron transport proteins get into the inner membrane. Return to Menu

Studies of yeast have helped us learn about the receptor and translocation machinery. One of the TOM's in yeast, called "MOM 19" works with MOM 72 to recognize and bind the proteins. In a recent paper by Harkness et al (J Cell Biology 124: 637-648, 1995), they created mutant yeast cells that included a defective gene for MOM19.

In this photo,  what is absent in the cells grown for 16 or 32 h in the drug?  When they did the assays for the proteins, what proteins would you predict would be missing? Tests showed that there was a dramatic decrease in most of the electron transport chain including cytochromes a/a3, and b. However, cytochrome C was unaffected. This suggests that another protein must control its import.