Topology of Prostaglandin H Synthase-1 in the Endoplasmic Reticulum Membrane

Authors: Ren Y.¹; Walker C.¹; Loose-Mitchell D.S.²; Deng J.¹; Ruan K.H.¹; Kulmacz R.J.¹

Source: Archives of Biochemistry and Biophysics, Volume 323, Number 1, October 1995 , pp. 205-214(10)

Publisher: Academic Press

Abstract:

Prostaglandin H synthase-1 is an integral endoplasmic reticulum membrane protein which catalyzes a key control step in prostaglandin biosynthesis. The overall arrangement of the prostaglandin H synthase-1 polypeptide with respect to the endoplasmic reticulum membrane was examined in transiently transfected COS-1 cells, using immunofluorescence microscopy. A bacterial toxin, streptolysin-O, was used for selective plasma membrane permeabilization and a detergent, saponin, for general membrane permeabilization. Treated cells were probed with six antibodies specific for particular prostaglandin H synthase-1 peptide segments and one antibody specific for an inserted viral reporter epitope. Control experiments established that actin, a cytoplasmic marker, was accessible to fluorescein-labeled phalloidin after streptolysin-O treatment, whereas antibodies against protein disulfide isomerase, an endoplasmic reticulum lumenal marker, bound only after saponin treatment. Using this approach to investigate prostaglandin H synthase-1, it was found that streptolysin-O treatment was sufficient to obtain staining of intracellular membranes by antibodies specific for the endogenous C-terminal segment, for the viral reporter inserted at the C-terminus, and for the protease-sensitive region near arg277. In contrast, saponin treatment was necessary for staining by antibodies specific for peptides spanning residues 51-66, 156-170, and 377-390. Antibodies targeted against residues 483-496 did not stain transfected cells even after saponin permeabilization, although they did bind to detergent-solubilized prostaglandin H synthase-1. These results indicate that the C-terminus and arg277 regions of the synthase can be exposed on the cytoplasmic side of the endoplasmic reticulum membrane, whereas regions near N-glycosylation sites are confined to the endoplasmic reticulum lumen and residues 483-496 are inaccessible from either side of the endoplasmic reticulum membrane.

Language: English

Document Type: Research article

Affiliations: 1: Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas, 77030 2: Deparment of Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas, 77030

Publication date: 1995-10-01

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• In this Subject: Anatomy & Physiology ,  Biology ,  Biochemistry
• By this author: Ren Y. ;  Walker C. ;  Loose-Mitchell D.S. ;  Deng J. ;  Ruan K.H. ;  Kulmacz R.J.

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