Signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p of the mitochondrial inner membrane protease

We have performed a site-directed mutagenesis study showing that residues c omprising the type I signal peptidase signature in the two catalytic subuni ts of the yeast inner membrane protease, Imp1p and Imp2p, are functionally important, consistent with the idea that these subunits contain a serine/ly sine catalytic dyad, Previous studies have shown that Imp1p cleaves signal peptides having asparagine at the -1 position, which deviates from the typi cal signal peptide possessing a small uncharged amino acid at this position , To determine whether asparagine is responsible for the nonoverlapping sub strate specificities exhibited by the inner membrane protease subunits, we have substituted asparagine with 19 amino acids in the Imp1p substrate i-cy tochrome (cyt) b(2). The resulting signal peptides containing alanine, seri ne, cysteine, leucine, and methionine can be cleaved efficiently by Imp1p, The remaining mutant signal peptides are cleaved inefficiently or not at al l. Surprisingly, none of the amino acid changes results in the recognition of i-cyt b(2) by Imp2p, whose natural substrate, i-cyt c(1), has alanine at the -1 position. The data demonstrate that (i) although the -1 residue is important in substrates recognized by Imp1p, signal peptides having standar d and nonstandard cleavage sites can be processed by Imp1p, and (ii) a -1 a sparagine does not govern the substrate specificity of the inner membrane p rotease subunits.