Figure 5. Raft association and transport of Gas1p in 4Δ.Lass5.

(A) 4Δ.Lass5 was grown in LM (His−, Met−), 4Δ.LAG1 in LM (−ura) containing galactose instead of glucose, YPK9 and lcb1-100 in YPD. All cells were grown at 30 °C except for lcb1-100, which was kept at 24 °C. Exponentially growing cells were lysed with glass beads and aliquots of lysate were solubilized in Triton X-100 for a membrane association assay (see the Materials and methods section). Triton X-100 lysates were subjected to ultracentrifugation, the soluble (SN) and the pellet (P) fractions were processed for Western blotting using anti-Gas1p (A) or anti-CPY antibodies (B), allowing the detection of mature (m) and precursor (p) forms. (A) Quantitation of results of top panel and of a second identical experiment is also shown. In (B) sec18Δ cells were incubated at 37 °C for 0, 1 and 3 h before lysis. For kinetic analysis of Gas1p (C) and CPY (D) maturation, yeast cells were metabolically labelled with [³⁵S]methionine and [³⁵S]cysteine, the label was chased for 0–60 min and Gas1p and CPY were immunoprecipitated from cell extracts, resolved by SDS/PAGE, and detected by phosphoimaging and by fluorography. Relative amounts of mature proteins are indicated in bottom panels.