HUMAN MITOCHONDRIAL IMPORT RECEPTOR, TOM20P - USE OF GLUTATHIONE TO REVEAL SPECIFIC INTERACTIONS BETWEEN TOM20-GLUTATHIONE S-TRANSFERASE AND MITOCHONDRIAL PRECURSOR PROTEINS

The cytosolic domain of the human mitochondrial protein import recepto r, hTom20, has been expressed as a fusion protein with glutathione S-t ransferase (GST) in bacteria and the purified protein immobilized on S epharose beads. To discriminate between specific binding of precursor proteins with the receptor and non-specific binding, precursors were r ecovered as a complex with GST-hTom20 following competitive elution fr om the beads with reduced glutathione. Here, we describe the specifici ty of this assay and demonstrate that the cytosolic domain of hTom20 i nteracts directly with the transcription-translation product of precur sor proteins that bear a diverse array of targeting signals. Such prot eins include a matrix protein (pODHFR), a polytopic integral protein o f the inner membrane (uncoupling protein), a beta-barrel protein of th e outer membrane (VDAC/porin) as well as bitopic integral proteins whi ch are inserted into the outer membrane by either an NH2-terminal or C OOH-terminal signal anchor sequence (yTom70(1-29)DHFR and Bcl-2, respe ctively). (C) 1997 Federation of European Biochemical Societies.