Figure 11.

Intracellular retention of insulin is perturbed in a point mutant changing the insulin B5 histidine to threonine. The strains are pep4Δ mutants in order to stabilize the intracellular protein. (A) Intracellularly retained and secreted insulin upon expression of the nonmutagenized ICFP control (Con) or the H90T-ICFP mutant (B5T) with a GAL1 promoter on a centromeric plasmid. Cells were pulse labeled for 30 min and chased for 60 min. The lysed cells and media underwent insulin immunoprecipitation before analysis by SDS-PAGE and fluorography. Note the dramatically increased secretion of the B5T mutant insulin (open arrows). Where indicated, the samples were digested with PNGaseF before SDS-PAGE. Only the insulin bands are shown in order to facilitate direct comparison of insulin secretion efficiency. C, cells; M, medium. (B) Intracellular nonmutagenized insulin control protein (Con) or the mutagenized insulin (B5T) followed by sucrose velocity gradient centrifugation analysis as in the legend to Fig. 10 A. Note that sedimentation of the B5T mutant (arrow) is clearly inhibited. U, top half of gradient; L, bottom half of gradient.