Figure 5.

Ssy5 cleavage of Stp2 is not impaired by charged amino acids at the P4 position. A, schematic presentation of Stp2 with expanded RII; the Ssy5 cleavage site, substrate P and P′ sites, as well as the corresponding S and S′ protease subsites are depicted (28). Amino acid substitutions at the P4, P1, and P′1 positions and summary of their effect on Ssy5 cleavage and growth as in B: red, impaired; green, neutral. B, Ssy5 cleavage of Stp2 (upper panels). Immunoblot analysis of protein extracts from strain CAY123 (stp1Δstp2Δ) carrying plasmids pAM059 (V90G), pAM060 (V90D), pAM063 (V90S), pAM064 (V90K), pAM061 (C93A), pAM062 (C93D), pAM069 (C93F), pAM068 (A94F), or pMB30 (WT). Immunoblots were developed with α-myc; the immunoreactive forms of N-terminal myc-tagged Stp2 are schematically depicted at their corresponding positions of migration. Growth of strain CAY123 (stp1Δstp2Δ) carrying plasmids as in the upper panel. 10-Fold dilutions of cultures grown in SD were spotted onto YPD and YPD+MM; plates were incubated at 30 °C for 2 days and photographed. Molecular markers (kDa) are indicated at the position of migration (left of immunoblots).