Figure 3.

Mutation of residues comprising the predicted hydrophobic patch on the surface of the catalytic domain. Ten positions mutated to correspond to their homologous residues in subtilisin Carlsberg (37) were studied. (A) Analysis of subcellular localization by fluorescence microscopy. (B) Western blotting using anti-GFP antibody. From the results of studies of subcellular localization and secretion, those mutants were classified into three groups: Golgi/secreted (I326K, S349E, and W413S), ER/nonsecreted (V292Y, T328D, L351E, and Q408Y), and cell death (apoptosis-inducing) (L410R, V412S, and F441K). In the cell death group, fragmentation of nuclei (arrows) was detected by nuclear staining using Hoe33342 (C) and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells were stained (dark brown) (D). In the latter experiment, a positive control for apoptosis was provided by treatment of WT PC3 with tunicamycin (Upper Middle). (Magnifications: A, ×800; C, ×400; D, ×200.)