TIM44, a peripheral inner membrane protein, tethers mitochondrial HSP70 to the import channel. A model peptide substrate rapidly released mitochondrial HSP70 from TIM44, even in the absence of ATP hydrolysis. In vivo, the analogous interaction of translocating polypeptide would release mitochondrial HSP70 from the channel.
TIMM44 is hydrophilic and contains no hydrophobic membrane-spanning segments. The N terminus has a coiled-coil motif and a matrix-targeting segment with 6 positively and no negatively charged residues. Western blot analysis showed that TIMM44 is present in membranes as well as matrix and is most likely a 45-kD peripheral membrane protein loosely associated with the inner membrane of mitochondria.