Translocation arrest of an intramitochondrial sorting signal next to Tim11 at the inner-membrane import site

The import of proteins from the cytosol into the mitochondrial matrix involves the concerted action of two separate import systems: the TOM system in the outer membrane, and the TIM system in the inner membrane. Here we report that the inner-membrane system also sorts proteins to the intermembrane space. Some intermembrane-space proteins, such as cytochromes b2 and c1, are synthesized with a complex pre-sequence consisting of a positively charged matrix targeting signal followed by an uncharged sequence that acts as sorting signal for the intermembrane space. We show that this sorting signal can be efficiently crosslinked to an inner-membrane protein of relative molecular mass 11K after the mature part of the precursor has been sorted to the intermembrane space. The 11K protein, which we term Tim11, is a component of the protein import system in the inner membrane.