FIGURE 2:

The internal MPP cleavage site in Arg5,6 is flanked by an iMTS-L and adheres to the R-2 motif. (A) Arg5,6 was subjected to TargetP profiling. High values indicate regions within the protein that structurally resemble mitochondrial presequences. (B) Putative cleavage sites for mitochondrial processing proteases in the second iMTS-L region of Arg5,6 were predicted with MitoFates. Mutations in the predicted recognition motif were introduced by site-directed mutagenesis. (C) Arg5,6 variants with and without the mutations displayed in B were synthesized as radiolabeled precursors and incubated with isolated mitochondria, followed by removal of nonimported material by PK treatment. Processing of the imported proteins was analyzed by SDS–PAGE, Western blotting, and autoradiography.