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

Substrate access channel topology in membrane-bound prostacyclin synthase

“…Crystallographic studies of detergent-solubilized [9] PGHS-1 and PGHS-2 and topological studies for membrane-bound [10,11] PGHS-1 and -2 suggest that the catalytic domains of the proteins lie on the luminal side of the ER, anchored to the membrane by hydrophobic side chains of amphipathic helices A-D. These hydrophobic side chains of the putative membraneanchor domains also form an entrance to the substrate-binding channel and potentially form an initial docking site for the lipid substrate, arachidonic acid [9,30].…”

“…PGI # biosynthesis presumably involves efficient co-ordination in the ER membrane between PGIS and prostaglandin H # synthase (PGHS), which produces PGH # . Results from crystallography and topology studies have indicated that the catalytic domain of PGHS is on the luminal side of the ER, with the cyclooxygenase substrate channel of PGHS immersed in the ER membrane [9][10][11]. The PGHS lipid substrate, arachidonic acid, thus appears to be delivered to the cyclo-oxygenase channel via the ER lipid bilayer.…”

“…Procedures used for selective permeabilization of cell membranes were described previously [10,20]. Briefly, ECV cells or transfected COS-1 cells grown on coverslips were washed with 25 mM Hepes, pH 7.4, containing 2.5 mM magnesium acetate, 25 mM KCl and 250 mM sucrose (Hepes buffer) and then incubated at 0 mC (for selective permeabilization of the plasma membrane) or 37 mC (for general permeabilization of plasma and ER membranes) for 10 min with 1.6 units\ml streptolysin O (SLO), which had been pre-activated with 4 mM dithiothreitol in Hepes buffer at 37 mC for 10 min.…”

“…SLO treatment at 0 mC was used to selectively permeabilize the plasma membrane without perturbing the ER membrane. SLO treatment at 37 mC was used to permeabilize the plasma and internal membranes and allow the antibodies to reach the luminal side of the ER membrane [10,20]. Triton X-100, a non-ionic detergent, was used to achieve maximal exposure of ER membrane proteins [20].…”

“…Triton X-100, a non-ionic detergent, was used to achieve maximal exposure of ER membrane proteins [20]. To confirm that the desired permeabilization was achieved, transfected COS-1 cells permeabilized with the various treatments described above were incubated with rhodamine-labelled phalloidin (targeted at actin in the cytoplasmic compartment) or with antibody to protein disulphideisomerase (PDI), which is a major ER luminal protein [10,32]. Cells permeabilized with SLO at 0 mC were stained by rhodaminelabelled phalloidin, but were not stained by antibody to PDI.…”