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 receptor, hTom20, has been expressed as a fusion protein with glutathione S-transferase (GST) in bacteria and the purified protein immobilized on Sepharose beads. To discriminate between specific binding of precursor proteins with the receptor and non-specific binding, precursors were recovered as a complex with GST-hTom20 following competitive elution from the beads with reduced glutathione. Here, we describe the specificity of this assay and demonstrate that the cytosolic domain of hTom20 interacts directly with the transcription-translation product of precursor proteins that bear a diverse array of targeting signals. Such proteins include a matrix protein (pODHFR), a polytopic integral protein of the inner membrane (uncoupling protein), a beta-barrel protein of the outer membrane (VDAC/porin) as well as bitopic integral proteins which are inserted into the outer membrane by either an NH2-terminal or COOH-terminal signal anchor sequence (yTom70(1-29)DHFR and Bcl-2, respectively).
Schleiff
E
E
Department of Biochemistry, McGill University, Montreal, Canada.
Shore
G C
GC
Goping
I S
IS
D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005982:Glutathione Transferase; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C084008:TOMM20 protein, human; D005982:Glutathione Transferase