Figure 5.

CPY sorting is complemented by Arf-binding domain mutants in yeast. (A) Pulse-chase analysis of newly synthesized CPY in wild-type, gga1gga2, and gga1gga2 harboring low copy plasmids of Ggas. Time course is indicated in minutes. Expression of Gga2p or Gga2p^I207N (right) fully complemented the CPY processing defect in gga1gga2 strains. Expression of Gga1p or Gga1p^L203Q (middle) partially complemented this defect. P1, unglycosylated endoplasmic reticulum form of CPY; P2, glycosylated Golgi form; M, fully processed mature (vacuolar) form. (B) Detection of secreted CPY by colony immunoblotting. Serial dilutions of yeast cultures were replica plated onto YPD plates. A signal on colony immunoblots indicates that CPY is secreted from the cell, suggesting that CPY is missorted. The CPY sorting defect of gga1gga2 strains (ΔΔ) was complemented by wild-type Gga2p and mutant Gga2p^I207N. Wild-type Gga1p partially complemented the CPY sorting defect, whereas mutant Gga1p^L203Q failed to complement.