Figure 2.

In wild-type yeast, ICFP is processed to single chain insulin but is not secreted efficiently. (A) Yeast expressing ICFP driven by a GAL1 promoter on a high copy plasmid were pulse labeled with [³⁵S]cysteine for the times indicated without further chase. (Note that in this strain the GAL1 promoter is not particularly strong.) Cells were lysed and insulin immunoprecipitated and digested with PNGaseF before nonreducing SDS-PAGE. Note that the initially synthesized precursor is ∼14 kD, which is processed to an ∼5-kD band comigrating with authentic insulin (INS). (B) Yeast expressing ICFP driven by a GAL1 promoter on a low copy (CEN) plasmid were pulse labeled for 10 min with [³⁵S]cysteine and either lysed or chased for a further 30 min in complete medium. Note that after chase, insulin-immunoprecipitable bands are undetectable in either the medium or cell lysate. C, cells; M, medium.