Glycosylation site binding protein (GSBP) has been shown to be identical to protein disulfide isomerase (PDI; EC 5.3.4 We developed an 125I-labeled photoaffinity probe based on the tripeptide Asn-Lys-Thr, the acceptor sequence for oligosaccharyl transferase. This enzyme of the endoplasmic reticulum (ER) catalyzes N-glycosylation of such peptides as well as nascent polypeptide chains (1). Photolysis of the photoaffinity probe in the presence of microsomes from a variety of higher eukaryotes resulted in the radiolabeling of a 57-kDa protein (2). Antibody against the 57-kDa protein was used to clone a cDNA encoding this lumenal ER protein (termed glycosylation site binding protein, GSBP; refs. 3 and 4). The deduced amino acid sequence revealed strong similarity (94% if conservative substitutions were allowed) with rat liver protein disulfide isomerase (PDI; EC 5.3.4.1), a lumenal ER protein that is thought to catalyze the formation and rearrangement of disulfide bonds in proteins (ref. 5; see ref. 6 for a review). The amino acid sequence of GSBP also was highly homologous to human thyroid hormone binding protein (THBP) and the human 18-subunit of prolyl hydroxylase (13-PH; refs. 7 and 8), both of which were found in the lumen of the ER. There is now evidence in higher eukaryotes that within a single species, THBP, PDI, and B3-PH are the same polypeptide (9) and that human THBP and rat PDI both exhibit GSBP activity (10). Further, it is clear that although rat liver PDI and GSBP are identical, the active sites for PDI activity and for binding the photoaffinity probe are separate and distinct (R. Noiva and W.J.L., unpublished observations).To better study the properties of this multifunctional lumenal protein, we turned to the yeast Saccharomyces cerevisiae because this organism contains a genetic system that affords easy access to various types of mutants. In this organism, in contrast to higher eukaryotes, the photoaffinity probe labeled a protein initially believed to have a molecular mass of 80 kDa (2) MATa, pho3, phoS, trpl, leu2, ura3, his3) and PDI mutational analyses (S. cerevisiae diploid W303; MATa/MATa, ade2/ade2, his3/his3, trpl/trpl, ura3/ura3, leu2/leu2, canl-100/canl-100). Routine recombinant DNA methodology and Escherichia coli colony hybridization were performed as described (11). Southern analysis was performed as described (12) with the Pvu I-Sal I fragment internal to the PDI gene as a hybridization probe. Endoglycosidase H (endo H) treatments were performed as described (13).Standard protocols (14) were followed for growth of yeast cells, media, transformation, sporulation, tetrad dissection, and isolation of genomic DNA. Yeast cells treated with tunicamycin (Sigma) were grown to mid-logarithmic phase, at which time the drug was added to a final concentration of 10 ,ug/ml. The strain was grown for 3 hr in the presence of the drug, after which protein extracts were prepared as described below.

Protein disulfide-isomerase (PDI), which reactivates inactive scrambled RNase, was purified from Saccharomyces cerevisiae. The enzyme was purified 1,850-fold to apparent homogeneity by five purification steps: 30-70% ammonium sulfate fractionation, DEAE Toyopearl-650S and Butyl Toyopearl-650S chromatographies, and differential Phenyl-5PW HPLC with or without cysteine. The native enzyme had an apparent Mr of 140,000 on gel filtration chromatography, and its NH2-terminal was blocked. The Mr of its subunits were estimated to be 70,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the enzyme is probably composed of two identical subunits. The Mr of the subunits changed to 60,000 on endoglucosaminidase H treatment, indicating that the enzyme is transported into the endoplasmic reticulum. The enzyme has a pH optimum of 8.5, and pI of 4.02. Its enzymic properties were compared with those of purified bovine liver PDI. The Km values of yeast and bovine PDIs for scrambled RNase were 1 x 10(-5) and 2 x 10(-5) M, and their Vmax values were 6 and 7 units/mg protein, respectively. The two enzymes showed no significant differences in Km or Vmax values with respect to thiol compounds. Bacitracin inhibited both PDIs in the same fashion. These results indicate that this yeast PDI corresponds to mammalian PDI.

MPD1, a yeast gene the overexpression of which suppresses the inviability caused by the loss of protein disulfide isomerase (PDI) was isolated and characterized. The MPD1 gene product retained a single disulfide isomerase active site sequence (APWCGHCK), an N-terminal putative signal sequence, and a C-terminal endoplasmic reticulum (ER) retention signal, and was a novel member of the PDI family. The gene product, identified in yeast extract, contained core size carbohydrates. MPD1 was not essential for growth, but overexpression of the gene suppressed the maturation defect of carboxypeptidase Y caused by PDH deletion, indicative of the related function to PDI in the yeast ER.