THE USE OF CHEMICAL CROSS-LINKING TO IDENTIFY PROTEINS THAT INTERACT WITH A MITOCHONDRIAL PRESEQUENCE

Previous work has shown that when yeast mitochondria are incubated in the presence of the presequence peptide pL4(1-22), the peptide is impo rted and accumulates within the mitochondrial membranes, presumably at the import sites. If the extramitochondrial concentration of peptide is sufficiently high, enough peptide accumulates within the import sit es to prevent the uptake of authentic precursor proteins. We have used chemical cross-linking to probe the interaction of this peptide with yeast mitochondrial proteins. We found that radiolabeled pL4(1-22) cou ld be reproducibly cross-linked to a number of polypeptides. Interesti ngly, nearly all were membrane proteins. Several of the cross-linked p roteins were located in the outer membrane, while others were located in the inner membrane, The interaction between the peptide and many of the cross-linked products was shown to be specific by two independent criteria, First, an excess of unlabeled peptide acted as a competitor in the cross-linking reaction, and, second, treatment of the peptide with the alkylating agent N-ethylmaleimide dramatically reduced its ab ility to form cross-links. Two of the cross-linked species corresponde d to the outer membrane proteins, Mas70p and ISP42. Significantly, bot h of these proteins have previously been shown to play critical roles in mitochondrial protein import. While the role of the other cross-lin ked proteins in the import process remains to be determined, the resul ts of this study demonstrate that our experimental approach may be use ful in identifying components of the import machinery as well as prote ins that interact with mitochondrial presequences.