Protease secretion in the sur7 Δ null mutant strain. (A) Extracellular protease secretion was assayed using a BSA degradation plate assay. Overnight cultures were spotted onto BSA plates and incubated at 30°C for 24 and 48 h. The relative amount of extracellular protease activity is indicated by the halo surrounding the fungal colony. (B) BSA degradation and Sap2p levels in liquid cultures were also assessed. Overnight cultures were shifted to medium containing BSA as the sole nitrogen source and incubated at 30°C for 6 hours. The degree of proteolysis of BSA was analyzed by reducing SDS-PAGE and Coomassie blue staining. Samples were normalized for loading with respect to culture density. Lanes containing standard protein markers (M) and intact BSA are shown for reference. (C) Cell-free supernatants prepared as described in (B) were analyzed by Western blotting using anti-Sap2p antibodies (from M. Monod). The triple deletion mutant strain sap1-3 Δ (from B. Hube) was used as a negative control. rSap2 indicates purified recombinant Sap2p (from M. Monod) used as a positive control.