FUNCTIONAL AND STRUCTURAL-PROPERTIES OF THE MITOCHONDRIAL OUTER-MEMBRANE RECEPTOR TOM20

Tom20 is an outer mitochondrial membrane protein that functions as a c omponent of the import receptor complex for cytoplasmically synthesize d mitochondrial precursor proteins. The human homologue, hTom20, consi sts of an N-terminal membrane anchor region predicted between aa5-25 a nd a soluble cytosolic domain from aa30 to 145. To analyze the propert ies 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 s olution containing two domains which are involved in different functio ns of the receptor. The N-terminal region is involved in membrane bind ing (aa30-60) and recognition of the cleavable matrix targeting signal s (aa50-90). In addition, we have demonstrated that the receptor recog nizes the or-helical state of the matrix targeting signal. The dissoci ation constant for this interaction in the presence of a detergent whi ch induces this secondary structure is 0.6 mu M, one-fifth the value i n the absence of detergent. In aqueous solution, the region between aa 30 and 60 is loosely folded and stabilized against proteolytic cleavag e by interaction with detergents or a matrix targeting signal. Our wor k further shows that the remainder of the cytosolic domain of hTom20, aa60-145, is a compactly folded globular domain containing a region (a a90-145) that is critical for the recognition of proteins bearing inte rnal signal sequences such as the uncoupling protein and porin.