Temperature-sensitive forms of large and small invertase in a mutant derived from a SUC1 strain of Saccharomyces cerevisiae.

Mutagenesis of the sucrose-fermenting (SUC1) Saccharomyces cerevisiae strain 4059-358D yielded an invertase-negative mutant (D10). Subsequent mutagenic treatment of D10 gave a sucrose-fermenting revertant (D10-ER1) that contained the same amount of large (mannoprotein) invertase as strain 4059-358D but only trace amounts of the smaller intracellular nonglycosylated enzyme. Limited genetic evidence indicated that the mutations in D10 and D10-ER1 are allelic to the SUC1 gene. The large invertases from D10-ER1 and 4059-358D were purified and compared. The two enzymes have similar specific activity and Km for sucrose, cross-react immunologically, and show the same subunit molecular weight after removal of the carbohydrate with endo-beta-N-acetylglucosaminidae H. They differ in that the large enzyme from the revertant is rapidly inactivated at 55 degrees C, whereas that from the parent is relatively stable at 65 degrees C. The small invertase in extracts of D10-ER1 is also heat sensitive as compared to the small enzyme from the original parent strain. The low level of small invertase in mutant D10-ER1 may reflect increased intracellular degradation of this heat-labile form. In several crosses of D10-ER1 with strains carrying the SUC1 or SUC3 genes, the temperature sensitivity of the large and small invertases and the low cellular level of small invertase appeared to cosegregate. These findings are evidence that SUC1 is a structural gene for invertase and that both large and small forms are encoded by a single gene. A detailed genetic analysis is presented in a companion paper.

Isolation and characterization of a yeast gene, MPD1, the overexpression of which suppresses inviability caused by protein disulfide isomerase depletion.

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 PDI1 deletion, indicative of the related function to PDI in the yeast ER.

Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth.

A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth.

Secretion of an active nonglycosylated form of the repressible acid phosphatase of Saccharomyces cerevisiae in the presence of tunicamycin at low temperatures.

The role of mannan chains in the formation and secretion of active acid phosphatase of yeast (Saccharomyces cerevisiae), a repressible cell surface mannoprotein, was studied in yeast protoplast systems by using tunicamycin at various temperatures. At 30 degrees C, tunicamycin-treated protoplasts did not produce active acid phosphatase; however, at 25 or 20 degrees C they formed and secreted active enzyme. This form of acid phosphatase gave 59-, 57-, and 55-kDa bands on SDS-PAGE which neither bound to concanavalin A Sepharose, nor changed in molecular weight upon treatment with endoglycosidase H, indicating that the peptides are nonglycosylated. The nonglycosylated form, like its glycosylated counterpart, is a dimer on the basis of gel permeation chromatography. The Km for para-nitrophenyl-phosphate and Ki for inorganic phosphate of both glycosylated and nonglycosylated acid phosphatases were almost the same. These results suggested that 1) the conformation of the nonglycosylated acid phosphatase secreted at low temperatures is probably identical with that of the glycosylated one, and 2) the conformation of acid phosphatase is very important for its secretion. The rate of intracellular transport of nonglycosylated acid phosphatase is about one-fourth that of the glycosylated enzyme, indicating that glycosylation facilitates the transport of acid phosphatase proteins.

Purification and characterization of yeast protein disulfide isomerase.

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.

Relationship of large and small invertases in Saccharomyces: mutant selectively deficient in small invertase.

A mutant strain of Saccharomyces cerevisiae (D10-ER1) has been isolated after a two-step mutagenesis of strain 4059-358D (SUC 1) using ethyl methane sulfonate. Cells of this new strain produced a level of total invertase equaling that of 4059 but contained only trace amounts of the small, internal, aglycan form of the enzyme (less than 0.1% of total in D10-ER1 compared with 6% in 4059). When D10.ER1 was crossed with an invertase-hyperproducing strain dgr3 (SUC3), progeny were isolated (HZ400-5A and HZ400-2C) in which levels of total invertase had at least quadrupled. The percentage of small invertase, however, remained insignificant. Levels of small invertase in strain HZ400-5A were determined by affinity chromatography on conconavalin A-Sepharose, gel permeation chromatography, and isopycnic centrifugation in CsCl. The large invertase of the SUC1 yeasts described here was found to contain a form apparently greater in size than the large invertase of the SUC2 strain FH4C; this probably reflects a higher content of carbohydrate. The overall results of this study do not support a direct structural relationship between large and small invertases. The implications on invertase biosynthesis and structure are discussed.

The nucleotide sequence of the yeast PHO5 gene: a putative precursor of repressible acid phosphatase contains a signal peptide.

The nucleotide sequence of the PHO5 gene of the yeast, Saccharomyces cerevisiae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terminal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotic signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PHO5 gene are also discussed.

Glycosylation site binding protein and protein disulfide isomerase are identical and essential for cell viability in yeast.

Glycosylation site binding protein (GSBP) has been shown to be identical to protein disulfide isomerase (PDI; EC 5.3.4.1) in a variety of multicellular organisms. We have utilized immunological and biochemical techniques to determine if GSBP and PDI are identical in yeast. Antiserum prepared against yeast GSBP identified in microsomes by its ability to be labeled with a peptide photoaffinity probe was found to recognize PDI purified from yeast. Moreover, this purified yeast PDI was found to be specifically labeled by the photoaffinity probe originally used to identify GSBP in a variety of eukaryotes. On the basis of these observations, we conclude that yeast GSBP and PDI are the same protein. The structure of the yeast PDI gene revealed a product with sequence similarity to higher eukaryotic PDI/GSBP. Disruption of this gene in yeast resulted in a recessive lethal mutation, indicating that PDI/GSBP is required for cell viability.

Thyroid hormone binding protein contains glycosylation site binding protein activity.

Several lines of evidence provided by other workers indicate that within the same species thyroid hormone binding protein, the beta-subunit of prolyl hydroxylase, and protein disulfide isomerase are the same protein. We sought to determine if glycosylation site binding protein, a lumenal protein of the endoplasmic reticulum, also has the same primary structure. To accomplish this the level of glycosylation site binding protein (GSBP) activity, measured by photolabeling with a glycosylation site peptide probe, was carried out in preparations of 3T3 cells and in E. coli transformed with human thyroid hormone binding protein cDNA. The results strongly support the idea that GSBP is identical to these other lumenal proteins of the endoplasmic reticulum.

Overproduction of Mpd2p suppresses the lethality of protein disulfide isomerase depletion in a CXXC sequence dependent manner.

The third multicopy suppressor gene of the PDI1 deletion from Saccharomyces cerevisiae, MPD2, was isolated and characterized. The MPD2 gene encodes a protein with a putative signal sequence, ER retention signal, and a disulfide isomerase active site like sequence. The amino acid sequence around the active site like sequence is similar to the thioredoxin-like domains of PDI and PDI related proteins, although the similarity is comparatively low. A delta-pdi1 strain over-producing Mpd2p showed slow growth and was sensitive to 1 mM dithiothreitol. Mpd2p can be detected in wild type cells and is a glycoprotein. Although the MPD2 gene was not essential for growth, overexpression of the gene partially restored the maturation defect of carboxypeptidase Y caused by the PDI1 deletion. Mutagenesis analysis revealed that Mpd2p can compensate for the loss of PDI with its CXXC sequence.