Supplemental Materials

This article contains the following supporting material:

• Figure S1 - Gas1p is normally localized at the cell surface in cwh43Δ cells.
  
  (A) The amount of Gas1p from various mutant cells was estimated by immunoblotting using an anti-Gas1p antibody. Equal numbers of wild-type (WT), cwh43Δ, per1Δ, and gup1Δ cells were cultured in YPAD and harvested. Secreted proteins were precipitated from the medium using trichloroacetic acid (10% final concentration). Cell lysates and secreted proteins were separated by SDS-PAGE and detected by immunoblotting using an anti-Gas1p antibody. Dpm1p was used as a control for ER resident proteins. (B) Localization of mRFP-Gas1p is similar in wild-type and cwh43Δ cells. Wild type (WT) and cwh43Δ cells introduced with pMF923 (pRS315-mRFP-GAS1) were examined by fluorescence microscopy. (C) Most of the Gas1p was associated with DRMs in cwh43Δ cells. Cells were disrupted with glass beads, extracted with Triton X-100, and subjected to Optiprep density gradient centrifugation. Six fractions were collected and analyzed by immunoblotting with antibodies against Gas1p, Pma1p, and Pho8p. Pma1p was used as a marker for DRM-associated proteins, and Pho8p was used as a marker for non-DRM proteins.