The Yeast Mitochondrial Protein Import Receptor Mas20p Binds Precursor Proteins through Electrostatic Interaction with the Positively Charged Presequence

doi:10.1074/jbc.270.10.5565

The Yeast Mitochondrial Protein Import Receptor Mas20p Binds Precursor Proteins through Electrostatic Interaction with the Positively Charged Presequence (*)

From the Biozentrum, University of Basel, CH-4056 Basel, Switzerland

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Abstract

Protein import into yeast mitochondria is mediated by the four outer membrane receptors Mas70p, Mas37p, Mas20p, and Mas22p. These receptors may function as two subcomplexes: a Mas37p/Mas70p heterodimer and an acidic complex consisting of Mas20p and Mas22p. To assess the relative contribution of these subcomplexes to precursor binding, we allowed different precursors to bind to the surface of deenergized mitochondria, then reenergized the mitochondria and measured the chase of the bound precursors into the organelles. Productive binding of several precursors with a positively charged amino-terminal matrix targeting sequence, such as SU9-DHFR, hsp60, and mitochondrial cpn10, was strongly inhibited by salt, by low concentrations of a mitochondrial presequence peptide, and by a deletion of Mas20p, but was independent of Mas37p/Mas70p. In contrast, productive binding of the ADP/ATP carrier was not inhibited by salt, the presequence peptide, or a deletion of Mas20p, but was strongly dependent on Mas37p/Mas70p. The precursors of alcohol dehydrogenase III and the Rieske iron-sulfur protein had binding properties between these two extremes. The productively bound precursor of cpn10 could be cross-linked to Mas20p. We conclude that Mas20p binds mitochondrial precursor proteins through electrostatic interactions with the positively charged presequence, whereas Mas37p/Mas70p may recognize some feature(s) of the mature part of precursor proteins.

Footnotes

↵^§ Recipient of a fellowship from the Boehringer Ingelheim Foundation.
↵^¶ Supported by a long term fellowship from the Human Frontiers Science Program Organization.
↵* This study was supported by grants from the Swiss National Science Foundation (3-26189.89) and the Human Capital and Mobility Program of the European Economic Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹S. Gratzer, T. Lithgow, R. E. Bauer, E. Lamping, F. Paltauf, S. D. Kohlwein, V. Haucke, T. Junne, G. Schatz, and M. Horst, submitted for publication.
↵²M. Horst and W. Oppliger, manuscript in preparation.
↵³ The abbreviations used are:

DHFR

dihydrofolate reductase

CCCP

carbonyl cyanide m-chlorophenylhydrazone

PAGE

polyacrylamide gel electrophoresis

AAC

ADP/ATP carrier

PMSF

phenylmethylsulfonyl fluoride.
↵⁴S. Rospert and G. Schatz, manuscript in preparation.
⁵V. Haucke, T. Lithgow, and G. Schatz, manuscript in preparation.
- Received October 21, 1994.
- Revision received December 8, 1994.