Domain structure and lipid interaction of recombinant yeast Tim44

Tim44 is an essential component of the machinery that mediates the transloc ation of nuclear-encoded proteins across the mitochondrial inner membrane. It functions as a membrane anchor for the ATP-driven protein import motor w hose other subunits are the mitochondrial 70-kDa heat-shock protein (mhsp70 ) and its nucleotide exchange factor, mGrpE. To understand how this motor i s anchored to the inner membrane, we have overexpressed Tim44 in Escherichi a coli and studied the properties of the pure protein and its interaction w ith model lipid membranes. Limited proteolysis and analytical ultracentrifu gation indicate that Tim44 is an elongated monomer with a stably folded C-t erminal domain, The protein binds strongly to liposomes composed of phospha tidylcholine and cardiolipin but only weakly to liposomes containing phosph atidylcholine alone. Studies with phospholipid monolayers suggest that Tim4 4 binds to phospholipids of the mitochondrial inner membrane both by electr ostatic interactions and by penetrating the polar head group region.