Fig 7.

Extraction of the Ywp1 propeptide from intact cells by use of SDS. Yeast cell culture supernatants (CS) were harvested and compared with sequential SDS extracts of whole cells. SDS-PAGE was followed by protein staining with Coomassie blue R-250. (A) Wild-type C. albicans strain SC5314 (+) compared with ywp1/ywp1 knockout strain 4L1 (Δ). All samples were ethanol precipitated and digested with PNGase F prior to electrophoresis. The Ywp1 propeptide (arrow) was extracted from intact cells by SDS at 70°C but not at 50°C or below. (B) The “C. albicans 70° SDS” lanes show that the migration of the band at 11 kDa depended upon removal of its N-glycan with PNGase F; otherwise, it migrated only into the stacking gel. A similar pattern was seen for C. krusei. (C) PNGase F digests of similar samples from C. dubliniensis and C. lusitaniae; again, most of the putative Ywp1 propeptide was extracted from intact cells by SDS only at temperatures above 50°C. M, markers with the indicated masses (in kDa).