Extended Data Fig. 4: Stp1, Stp2, Stp3, Stp4 are secreted whereas Stp5 and Stp6 localize to the fungal plasma membrane upon constitutive expression.
From: A cell surface-exposed protein complex with an essential virulence function in Ustilago maydis
a-d, HA-specific western blot to reveal secretion of Stp proteins after constitutive expression from the otef promoter in AB33 (a), SG200Δkex2 (b) or SG200 (c, d). Supernatant fractions (S), as well as pellet fractions (P) were loaded. Cultures in (d) were grown in the presence of protease inhibitors. Stp1 contains several predicted Kex2 processing sites (Supplementary Fig. 1) and to detect Stp1-HA in culture supernatants it was necessary to express the protein in a strain lacking kex2. The tubulin specific western blot serves as a lysis control. Proteins of the expected size are marked with an asterisk. The molecular weight in kDa is indicated. e, HA-specific western blot revealing membrane association of HA-Stp5 and Stp6-HA. Total extract (T), plasma membrane fraction (M) and soluble fraction (Sol) from SG200-derived strains expressing the indicated fusion proteins were loaded. mCherrycyt serves as control for a cytoplasmic protein, Cmu1-HA serves as control for a secreted protein5 and Pit1-HA serves as control for a fungal plasma membrane protein57. Full-length proteins are marked with an asterisk and molecular weight markers are indicated. HA-Stp6 co-migrates with an unspecific signal also detected in the pellet fraction of SG200 in (d).
