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.
Highlights
The relative contributions of these different receptor subunits to the overall import process have not been firmly established
Mas37pIMas70p promotes the import of the ADP/ATP carrier, cytochrome c1, the F I-ATPase l3-subunit, and the mitochondrial isozyme III of the alcohol dehydrogenase, but not import of artificial precursors containing dihydrofolate reductase (DHFR)3 as a passenger protein
Experiments in which the presequences of authentic precursors were fused to DHFR have suggested that Mas20p/Mas22p, through their cytosolically exposed "acid bristles," may bind the basic and amphiphilic mitochondrial presequences [9, 12]
Summary
5565-5570, 1995 Printed in U.S.A. The Yeast Mitochondrial Protein Import Receptor Mas20p Binds Precursor Proteins through Electrostatic Interaction with the Positively Charged Presequence*. Only Mas22p is essential for protein import and viability of yeast; Mas20p, Mas37p, and Mas70p can be deleted singly or in pairwise combination without blocking import as long as the level of Mas22p is maintained [9] It is still unknown how the import receptors recognize precursor proteins destined for mitochondria. We found that the NHz-terminal basic and amphiphilic presequences are bound by the acid bristle complex containing Mas20p and Mas22p, that this binding is blocked by a chemically synthesized presequence peptide, and that it is predominantly mediated by a salt-sensitive electrostatic interaction. Binding of precursors to Mas37pl Mas70p is only partly inhibited by high salt or by a presequence peptide, confirming that this receptor subcomplex recognizes determinants in the mature part of precursor proteins.
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