Fig. 3.

The uncoupling mutation E230A in Gef1 abolishes maturation of multicopper oxidases. (A) Total membrane protein isolated from the indicated strains resolved on a clear native gel and incubated with PPD to reveal multicopper oxidase activity (upper panel). After the in-gel assay, the gel was stained with Coomassie Blue to demonstrate equal loading of all lanes. Purified human ceruloplasmin was loaded as a positive control for multicopper oxidase activity. (B) Fet3GFP expression analysed in the indicated strains to verify expression of Fet3 in the strains lacking Gef1 antiporter activity. (C) 4PC-Gef1 expressed under the control of the endogenous GEF1 promoter to monitor physiological changes of Gef1 expression under iron starvation. Expression was analysed by western blotting of total cell lysates.