Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking

Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway, Amino acids with benzophenone side chains of various lengths [DL-2-amino-3-(p-benzoylphenyl)propanoic acid (1) and DL-2-amino-5-(p-benzoylphenyl)pentanoic acid (2)] were incorporated at specific sites throughout the cytochrome b(2)-dihydrofolate reductase fusion proteins, pb(2)(220)-DHFR and pb(2) Delta 19(220)DHFR, which were destined for the intermembrane space and the matrix in mitochondria, respectively, In vitro import of pb(2)(220)-DHFR and pb(2) Delta 19(220)-DHFR bearing 1 or 2 into isolated yeast mitochondria was arrested so that the N terminus reached the intermembrane space or the matrix, respectively, while the DHFR domain remained at the mitochondrial surface. The matrix-targeted pb(2) Delta 19(220)-DHFR was photocrosslinked to Tom40 in the outer membrane, Tim44 in the inner membrane, and Ssc1p in the matrix, suggesting that the protein has an extended conformation in the import channels, On the other hand, incorporation of 2 at various positions in the 50-residue segment of intermembrane-space-targeted pb(2)(220)-DHFR gave photocrosslinks only to Tom40, suggesting that the segment is not in an extended conformation, but localized near Tom40. The N-terminal portion of pb(2)(220)-DHFR, but not pb(2) Delta 19(220)-DHFR, was photocrosslinked to an as-yet-unidentified mitochondrial component to generate a 95-kDa crosslinked product.