The role of ERO1 in oxidative protein folding in the endoplasmic reticulum

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1999.Includes bibliographical references.The formation of native disulfide bonds is critical for the folding and stability of many secreted proteins. We describe an essential S. cerevisiae gene, ER01, which encodes a conserved ER membrane protein required for disulfide bond formation in the er .doplasmic reticulum (ER). In a conditional ero 1-1 mutant, secretory proteins that would normally contain disulfide bonds, such as carboxypeptidase Y (CPY), are retained in the ER in reduced form, as shown by thiol modification with AMS. ER01 levels determine cellular oxidizing capacity, since mutation of ER01 causes hypersensitivil/ to the reductant OTT, whereas overexpression of ER01 confers resistance to OTT. Moreover, the thiol oxidant diamide can restore growth and secretion to ero1 mutants. These results suggest that Ero1p provides the oxidizing equivalents utilized for disulfide bond formation in the ER. Oxidizing equivalents are transferred directly from Ero1p to the abundant ER oxidoreductase PDI (protein disulfide isomerase) and its homolog Mpd2p. PDI is oxidized in wild-type cells, but reduced in the ero 1-1 mutant. Thiol-disulfide exchange between POI and Ero1p is indicated by the capture of PD1-Ero1p mixed-disulfides. PDI oxidizes secretory proteins, since newly-synthesized CPY remains fully reduced in POI-depleted cells. Mixed-disulfides between PDI and p1 CPY are also detected, indicating that PDI engages directly in thiol-disulfide exchange with this substrate. Together, these results define a pathway for protein disulfide bond formation in the ER wherein oxidizing equivalents flow from Ero1p to POI (and Mpd2p) and then to substrate proteins through direct thiol-disulfide exchange reactions. Oxidized glutathione (GSSG) does not serve as an obligate intermediate In this pathway, since oxidative protein folding proceeds normally in a gsh 1.1 mutant devoid of intracellular glutathione. Mutational analysis of ER01 identifies two pairs of conserved, vlclnal cystelnes essential for Ero1p function. Mutation of Cys100, Cys105, Cys352, or Cys355 of Ero1 p disrupts cell viability, CPY folding, and thiol-disulfide exchange between POI and Ero1p. Cys100 of Ero1p may be preferentially attacked by POI, while the Cys352- Cys355 disulfide may re-oxidize the Cys 100-Cys 105 cystelne pair. The properties of yeast Ero1 p resemble those of E. coli DsbB.by Alison R. Frand.Ph.D