INTERACTIONS OF THE HUMAN MITOCHONDRIAL PROTEIN IMPORT RECEPTOR, HTOM20, WITH PRECURSOR PROTEINS IN-VITRO REVEAL PLEIOTROPIC SPECIFICITIES AND DIFFERENT RECEPTOR DOMAIN REQUIREMENTS

Tom20 is part of a multiple component, dynamic complex that functions to import specific cytosolic proteins into or through the outer membra ne of the mitochondrion, To analyze the contribution of Tom20 to precu rsor protein recognition, the cytosolic domain of the human mitochondr ial import receptor, hTom20, has been expressed as a fusion protein wi th glutathione S-transferase and conditions established to measure spe cific interactions of the receptor component with precursor proteins i n vitro, Reconstitution of receptor binding from purified components r evealed that a prototypic matrix-destined precursor protein, pODHFR, i nteracts with Tom20 by a mechanism that is dependent on an active matr ix targeting signal but does not require cytosolic components or ATP, Binding was influenced by both salt concentration and detergent, The e ffect of salt or detergent, however, varied for different precursor pr oteins, In particular, detergent selectively enhanced binding of pODHF R to receptor, possibly because of induced changes in the structure of the signal sequence, Finally, mutations were introduced into hTom20 w hich had a dramatic effect on binding of some precursor proteins but n ot on others, Taken together, the results suggest that; hTom20 recogni zes and physically interacts with precursor proteins bearing a diverse array of topogenic sequences and that such pleiotropic specificity fo r these precursor proteins may involve different domains within the re ceptor molecule.