The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.In eukaryotes, the specific folding of proteins targeted to the secretory pathway or to extracellular space occurs in the endoplasmic reticulum (ER). For many secretory proteins, the proper folding requires the formation of intra-and intermolecular disulfide bonds (for reviews, see references 15, 21, and 39). The pathway of oxidative protein folding has been extensively studied in Saccharomyces cerevisiae. Genes have been identified that are involved in redox homeostasis within the ER. The protein folding process requires numerous chaperones and enzymes. The core pathway contains two conserved proteins: Pdi1p and Ero1p. Protein disulfide isomerase (PDI) catalyzes formation, isomerization, and reduction of disulfide bonds of substrate proteins (18,22,23,33). The ER membrane-associated protein Ero1p (ER oxidoreduction) introduces oxidizing equivalents through a flavin-dependent mechanism, engaging thiol-disulfide exchange with Pdi1p (17,38). Mutations in ERO1 and PDI1 result in cells that are sensitive to the reducing agent dithiothreitol (DTT) and that accumulate proteins that normally contain disulfide bonds in reduced form in the ER. The accumulation of reduced proteins induces the unfolded protein response (16,19,29,30). Overexpression of ERO1 results in cells resistant to DTT (16). A few other proteins functionally related to Pdi1p or to Ero1p have also been identified in S. cerevisiae. Overproduction of Mpd1, Mpd2, Eug1, and Eps1 can partially complement the loss of Pdi1p, and overproduction of Erv2 partially complements the loss of Ero1. Unlike ERO1 a...