Figure 4.

Mutation of Cys100 of Ero1p blocks oxidation of Pdi1p in vivo. The in vivo oxidation state of Pdi1p was assessed by the modification of free thiols with AMS after the precipitation of cellular proteins with TCA. Samples were resolved by nonreducing SDS-PAGE and Pdi1p detected by immunoblotting. Samples were prepared from cells with a chromosomal deletion of ERO1 that expressed either ero1-A100-myc (CKY686) or ERO1-myc (CKY600). To provide a standard for the mobility of reduced Pdi1p, one sample of CKY600 was treated with 10 mM DTT. The AMS-modified reduced and oxidized forms of Pdi1p are indicated.