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

Abstract

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active sites for PDI activity and for binding the photoaffinity probe are separate and distinct (R. Noiva and W.J.L., unpublished observations).To better study the properties of this multifunctional lumenal protein, we turned to the yeast Saccharomyces cerevisiae because this organism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.

The protein disulphide-isomerase family: unravelling a string of folds

“…PDI is essential for the viability of S. cere isiae [15,[75][76][77][78], and its essential role is in the isomerization of disulphide bonds [16,38], although its redox activity is clearly important for substrates such as carboxypeptidase Y [79]. Mammalian PDI can rescue PDI-deficient yeast, despite only low sequence identity (30 %) between the two proteins [81].…”

The endoplasmic reticulum as a protein-folding compartment

“…The fulMength polypeptides released from ribosomes continue to fold and acquire intrachain disulphide bonds. Folding intermediates of many proteins associate with BiP/ GRP78 during the post-translational phase of folding23, 24 Post-translational folding usually takes only a few minutes, but sometimes it can go on for much longer. Initial folding of individual glycopolypeptide chains is often followed by oligomerization 37.…”

The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane

“…The ER-luminal enzyme and chaperone PDI is the predominant binding partner in the ER lumen for glycosylation acceptor peptides and unglycosylated peptides irrespective of their thiol content (34)(35)(36). PDI is composed of four thioredoxin modules, two enzymatically active ones (a, aЈ) and two inactive ones (b, bЈ) (37).…”