Cys100, Cys105, Cys352, and Cys355 of Ero1p are important for cell viability and for oxidative protein folding in the ER. (A) Complementation of the temperature-sensitive ero1-1 mutant by alanine substitution mutants of ERO1-myc. Cells were plated on rich medium and incubated overnight at restrictive temperature (38°C). Alleles of ERO1-myc with detectable rescuing activity were expressed in an ire1Δ genetic background to prevent induction of ERO1 by the UPR. Transformants of CKY605 (ero1-1 ire1Δ::URA3) carried the following plasmids: wild type (WT), pAF85 [CEN ERO1-myc]; 90, pAF153 [CEN ero1-A90-myc]; 100, pAF154 [CEN ero1-A100-myc]; 208, pAF155 [CEN ero1-A208-myc]; or 349, pAF156 [CEN ero1-A349-myc]. Transformants of CKY559 (ero1-1) hosted the following: 105, pAF122 [CEN ero1-A105-myc]; 352, pAF96 [CEN ero1-A352-myc]; 355, pAF95 [CEN ero1-A355-myc]; or vector, pRS316 [CEN URA3]. (B) Assays for the DTT sensitivity of cells with a chromosomal deletion of ERO1 rescued by an alanine substitution mutant of ERO1-myc. Three lawns of each strain were plated on rich medium and 10 μl of 3 M DTT applied to each lawn in a filter disk. The average diameter of the zone of growth inhibition (mm) was measured after incubation at 30°C for 1.5 days. The strains shown are as follows: WT, CKY600 (ero1-Δ1-500::LEU2 p[ERO1-myc]); 90, CKY601 (ero1-Δ1-500::LEU2 p[ero1-A90-myc]); 100, CKY602 (ero1-Δ1-500::LEU2 p[ero1-A100-myc]); 208, CKY603 (ero1-Δ1-500::LEU2 p[ero1-A208-myc]); 349, CKY604 (ero1-Δ1-500::LEU2 p[ero1-A349-myc]); and ero1-1, CKY559. (C) Processing of newly synthesized CPY in ero1-1 cells expressing each alanine substitution mutant of ERO1. Cells were pulse-labeled with [³⁵S]methionine and cysteine at 38°C for 20 min. CPY was immunoprecipitated and the samples resolved by SDS-PAGE. The ER (p1) and vacuolar (mature) forms of CPY are indicated. Samples were prepared from the same strains as in A, except that “— ” refers to untransformed CKY559.

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.

Model for the catalytic mechanism of Ero1p. Two redox-active disulfide bonds are present in Ero1p (upper left). The Cys100-Cys105 disulfide engages in thiol–disulfide exchange with Pdi1p, whereas the Cys352–Cys355 disulfide serves to reoxidize the Cys100–Cys105 cysteine pair, possibly through an intramolecular thiol–disulfide exchange reaction. Reoxidation of Ero1p could proceed via oxidation of the Cys352–Cys355 cysteine pair by an as yet unidentified electron acceptor. Only one thioredoxin-like domain of Pdi1p is shown.

Conserved cysteines in Ero1. (A) Seven cysteines are absolutely conserved among the eukaryotic sequence homologues of Ero1. These residues are at positions 90, 100, 105, 208, 349, 352, and 355 of the primary amino acid sequence of yeast Ero1p. The N-terminal hydrophobic sequence of Ero1p (shaded) is likely to serve as a membrane anchor or signal sequence. (B) Predicted amino acid sequences of the homologues of Ero1. The regions shown correspond to residues 90–107 and 347–357 of yeast Ero1p. ^aThe Candida albicans sequence is derived from an unfinished genome (http://www.ncbi.nlm.nih.gov/Microb_blast/unfinishedgenome.html). ^bIn the Caenorhabditis elegans genome, two adjacent sequences are predicted to specify proteins homologous to the N- and C-terminal portions of yeast Ero1p. It remains to be determined whether these sequences correspond to one or two genes.

The capture of mixed-disulfide complexes between each alanine substitution mutant of Ero1p-myc and CGHS–CGHS Pdi1p or CQHA Mpdp2. Cells overproducing a single alanine-substitution mutant of Ero1p-myc in addition to CGHS–CGHS Pdi1p were labeled with [³⁵S]methionine and cysteine and then suspended in 10% TCA to block further thiol–disulfide exchange in vivo. Ero1p-Pdi1p mixed disulfides were isolated by modifying free thiols with NEM prior to immunoprecipitation with anti-myc antibody under nonreducing but denaturing conditions. The primary immunoprecipitates were reduced with 100 mM DTT prior to reimmunoprecipitation with either anti-myc (1× loading) or anti-Pdi1p (7.5× loading) antibody. Samples were resolved by SDS-PAGE and analyzed with a 445si phosphorimager. The efficiency of mixed-disulfide capture is expressed as the ratio of the band intensity of reimmunoprecipitated Pdi1p to that of reimmunoprecipitated Ero1p-myc (per OD₆₀₀ U of extract), normalized to the value obtained with wild-type Ero1p-myc. (A) Strains derived from CKY598 (ero1-1 GAL2) were grown in SMM Raf/Gal and labeled at restrictive temperature (38°C). (B) Otherwise isogenic strains derived from CKY263 (GAL2) were labeled at 30°C. The strains shown were transformed with pAF132 [P_GAL1-pdi1-1966] in addition to the following: WT, pAF89 [2μ ERO1-myc]; 90, pAF124 [2μ ero1-A90-myc]; 100, pAF125 [2μ ero1-A100-myc]; 105, pAF126 [2μ ero1-A105-myc]; 208, pAF127 [2μ ero1-A208-myc]; 349, pAF128 [2μ ero1-A349-myc]; 352, pAF129 [2μ ero1-A352-myc]; or 355, pAF130 [2μ ero1-A355-myc]. (C) Mixed disulfides between CQHA Mpd2p and each alanine substitution mutant of Ero1p-myc were isolated as described above from wild-type cells (CKY263) transformed with pAF123 [P_GAL1-mpd2p-CQHA] instead of pAF132, and the complexes resolved directly by nonreducing SDS-PAGE. The efficiency of mixed-disulfide capture is expressed as the ratio of the band intensity of the Mpd2p-Ero1p-myc complexes to free Ero1p-myc, normalized to the value obtained with wild-type Ero1p-myc.

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.