THE ORIENTATION OF PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-1 AND SYNTHASE-2 IN THE ENDOPLASMIC-RETICULUM

Prostaglandin endoperoxide H synthases (PGHS)-1 and -2 are integral me mbrane 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 treat ment with membrane-selective permeants. With serum-stimulated, murine NIH/3T3 cells expressing PGHS-2, an anti-peptide antibody directed aga inst a domain near the COOH terminus of this isozyme caused staining o nly after all membranes were permeabilized with 0.2% saponin; no stain ing occurred with 3T3 cells treated with digitonin to permeabilize onl y the plasma membrane. Similarly, cos-1 cells expressing ovine PGHS-1 were stained with anti-peptide antibodies directed against (a) the ami no terminus (residues 25-35), (b) a domain containing the tryptic clea vage site at Arg(277) (residues 272-284), or (c) a region near the car boxyl terminus (residues 583-594) following permeabilization with sapo nin but not with digitonin or streptolysin O. The results obtained wit h the antibodies against the Arg(277)-containing domain of PGHS-1 were surprising because the enzyme is susceptible to tryptic cleavage at A rg(277) in microsomal preparations. However, enzymatic and immunochemi cal analyses of microsomes prepared from ovine vesicular glands and co s-1 cells indicated that these microsomes are not intact. Accordingly, our results indicate that the trypsin cleavage site (Arg(277)) as wel l as the NH2 and COOH termini of ovine PGHS-1 are on the luminal side of the ER. The NH2 terminus, the Arg(277) domain, and the N glycosylat ion sites of ovine PGHS-1 are part of a large soluble, globular struct ure 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 anal ogy, the highly homologous PGHS-2 are luminal ER proteins. Assuming th at the PGHS-1 and PGHS-2 present in the ER are functional in intact ce lls, our results indicate that PGH(2) synthesis from arachidonate occu rs in the lumen of the ER.