Limited proteolysis of Ppt1p and its effect on Ppt1p activity. (A) Recombinant Ppt1p samples trypsinized in the absence or presence of 250 μM AA were resolved by electrophoresis on 15% SDS-polyacrylamide gel and visualized by Coomassie staining. The length of digest (minutes) and the migration of molecular weight standards are indicated. (B) N-terminal sequence analysis of 45 kDa fragments shown in (A) was performed to identify the tryptic cleavage sites within the linker region between the TPR and catalytic domains. Tryptic cleavage sites are indicated with the inverted triangles. Bold underlined residues are conserved in both Ppt1p and PP5. (C) Recombinant Ppt1p was digested using either trypsin (T) or subtilisin (S) for 5 minutes in the absence of AA and assayed using either 32P-casein or 32P-MBP in the absence of AA. Data are presented as activity relative to control, which is the activity of full-length Ppt1p subjected to mock digest and assayed in the absence of AA. Results represent the mean ± S.E. from three or more independent experiments.