Functional and Structural Properties of the Mitochondrial Outer Membrane Receptor Tom20

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Abstract
Tom20 is an outer mitochondrial membrane protein that functions as a component of the import receptor complex for cytoplasmically synthesized mitochondrial precursor proteins. The human homologue, hTom20, consists of an N-terminal membrane anchor region predicted between aa5−25 and a soluble cytosolic domain from aa30 to 145. To analyze the properties of hTom20, we have expressed several truncations of the cytosolic domain as fusion proteins with glutathione S-transferase. Our studies reveal that the cytosolic region of hTom20 is a monomeric protein in solution containing two domains which are involved in different functions of the receptor. The N-terminal region is involved in membrane binding (aa30−60) and recognition of the cleavable matrix targeting signals (aa50−90). In addition, we have demonstrated that the receptor recognizes the α-helical state of the matrix targeting signal. The dissociation constant for this interaction in the presence of a detergent which induces this secondary structure is 0.6 μM, one-fifth the value in the absence of detergent. In aqueous solution, the region between aa30 and 60 is loosely folded and stabilized against proteolytic cleavage by interaction with detergents or a matrix targeting signal. Our work further shows that the remainder of the cytosolic domain of hTom20, aa60−145, is a compactly folded globular domain containing a region (aa90−145) that is critical for the recognition of proteins bearing internal signal sequences such as the uncoupling protein and porin.