title: The orientation of prostaglandin endoperoxide synthases-1 and -2 in the endoplasmic reticulum.
abstract: Prostaglandin endoperoxide H synthases (PGHS)-1 and -2 are integral membrane proteins of the endoplasmic reticulum (ER). The luminal versus cytoplasmic orientations of several epitopes of PGHS-1 and PGHS-2 were determined by immunocytofluorescent staining of cells following treatment with membrane-selective permeants. With serum-stimulated, murine NIH/3T3 cells expressing PGHS-2, an anti-peptide antibody directed against a domain near the COOH terminus of this isozyme caused staining only after all membranes were permeabilized with 0.2% saponin; no staining occurred with 3T3 cells treated with digitonin to permeabilize only the plasma membrane. Similarly, cos-1 cells expressing ovine PGHS-1 were stained with anti-peptide antibodies directed against (a) the amino terminus (residues 25-35), (b) a domain containing the tryptic cleavage site at Arg277 (residues 272-284), or (c) a region near the carboxyl terminus (residues 583-594) following permeabilization with saponin but not with digitonin or streptolysin O. The results obtained with the antibodies against the Arg277-containing domain of PGHS-1 were surprising because the enzyme is susceptible to tryptic cleavage at Arg277 in microsomal preparations. However, enzymatic and immunochemical analyses of microsomes prepared from ovine vesicular glands and cos-1 cells indicated that these microsomes are not intact. Accordingly, our results indicate that the trypsin cleavage site (Arg277) as well as the NH2 and COOH termini of ovine PGHS-1 are on the luminal side of the ER. The NH2 terminus, the Arg277 domain, and the N-glycosylation sites of ovine PGHS-1 are part of a large soluble, globular structure in crystalline ovine PGHS-1 (Picot, D., Loll, P. J., and Garavito, M. (1994) Nature, 367, 243-249). We conclude that PGHS-1 and, by analogy, the highly homologous PGHS-2 are luminal ER proteins. Assuming that the PGHS-1 and PGHS-2 present in the ER are functional in intact cells, our results indicate that PGH2 synthesis from arachidonate occurs in the lumen of the ER.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing 48824.
authors.forename: J C
authors.initials: JC
authors.lastname: Otto
authors.forename: W L
authors.initials: WL
authors.lastname: Smith
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry*; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry*; D011487:Protein Conformation; D012756:Sheep; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011451:Prostaglandin-Endoperoxide Synthases; D014357:Trypsin