Figure 5.

Cys352 and Cys355 are required for oxidation of Ero1p in vivo. The CA352, CA355, CA100, and CA105 mutants of Ero1p-myc were overproduced in wild-type cells (CKY263), and the in vivo oxidation state of these proteins assessed by the modification of free thiols with AMS after the TCA-precipitation of cellular proteins. Samples were resolved by nonreducing SDS-PAGE and Ero1p-myc detected by immunoblotting. To provide standards for the mobility of the reduced and oxidized forms of Ero1p, cells expressing wild-type Ero1p-myc were treated with 5 mM DTT, or not treated. Lanes are labeled by the position of the alanine substitution in Ero1p-myc.