Interactions of the Human Mitochondrial Protein Import Receptor, hTom20, with Precursor Proteins in Vitro Reveal Pleiotropic Specificities and Different Receptor Domain Requirements*
Abstract
Tom20 is part of a multiple component, dynamic complex that functions to import specific cytosolic proteins into or through the outer membrane of the mitochondrion. To analyze the contribution of Tom20 to precursor protein recognition, the cytosolic domain of the human mitochondrial import receptor, hTom20, has been expressed as a fusion protein with glutathioneS-transferase and conditions established to measure specific interactions of the receptor component with precursor proteinsin vitro. Reconstitution of receptor binding from purified components revealed that a prototypic matrix-destined precursor protein, pODHFR, interacts with Tom20 by a mechanism that is dependent on an active matrix targeting signal but does not require cytosolic components or ATP. Binding was influenced by both salt concentration and detergent. The effect of salt or detergent, however, varied for different precursor proteins. In particular, detergent selectively enhanced binding of pODHFR to receptor, possibly because of induced changes in the structure of the signal sequence. Finally, mutations were introduced into hTom20 which had a dramatic effect on binding of some precursor proteins but not on others. Taken together, the results suggest that hTom20 recognizes and physically interacts with precursor proteins bearing a diverse array of topogenic sequences and that such pleiotropic specificity for these precursor proteins may involve different domains within the receptor molecule.
Footnotes
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↵* This study was financed by operating grants from the Medical Research Council and the National Cancer Institute of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Recipient of a Fellowship of the HSP/II program from the German Academic Association Service (DAAD).
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↵§ To whom correspondence should be addressed: Dept. of Biochemistry, McIntyre Medical Science Bldg., McGill University, Montreal H3G 1Y6, Canada. Tel.: 1-514-398-7282; Fax: 1-514-398-7384; E-mail:shore{at}medcor.mcgill.ca.
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↵1 The abbreviations used are: UCP, uncoupling protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; DHFR, dihydrofolate reductase; MTS, matrix targeting signal; NEM, N-ethylmaleimide; VDAC, voltage-dependent anion channel.
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↵2 E. Schleiff, unpublished observations.
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- Received January 10, 1997.
- Revision received March 18, 1997.
