Figure 3.

Construction of HA- or mRFP-tagged Gas1p and Gas1^*p. (A) Schematic construction of HA-tagged and mRFP-tagged Gas1^*p. A 3× HA epitope tag or mRFP sequence was inserted into Gas1p after the secretion signal sequence. (B) Tagged Gas1 proteins are functional and complement the calcofluor white (CFW) sensitivity of gas1Δ. Isogenic W303-1A (WT), MFY156 (gas1Δ), MFY182 (gas1Δ HA-GAS1), and MFY183 (gas1Δ mRFP-GAS1) cells were spotted on plates containing YPAD or YPAD with 5 μg/ml CFW and then incubated at 30°C for 2 d. (C) HA-Gas1p behaves like the native form of Gas1p, but the mutant of HA-Gas1p is unstable and remains as the ER form. MFY207 (HA-GAS1) and MFY208 (HA-gas1^*) cells were grown overnight to mid-log phase. Equal cell numbers were disrupted, and total protein extracts were prepared and analyzed by immunoblotting using anti-HA and anti-mouse HRP-conjugated IgG, or anti-Dpm1p and anti-mouse HRP-conjugated IgG. ER and Golgi forms of HA-tagged Gas1p are shown by arrowheads. (D) The misfolded Gas1^*p is modified by the GPI anchor. MFY207 (HA-GAS1) and MFY208 (HA-gas1^*) cells were grown to a mid-log phase and labeled with myo-[1,2-³H]inositol for 3 h. Gas1p and Gas1^*p in cell lysates were then immunoprecipitated with anti-HA-agarose and analyzed by SDS-PAGE, followed by image analysis with a Molecular Imager. The ER and Golgi forms are indicated by arrows.