The membrane association sequences of the prostaglandin endoperoxide synthases-1 and -2 isozymes.
Based on the crystal structures of the prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and PGHS-2), four short amphipathic helices near the amino termini of these proteins have been proposed to act as membrane binding domains. We constructed a series of plasmids coding for amino-terminal sequences of the PGHS-1 and PGHS-2 joined to the green fluorescent protein from Aequorea victoria, and we examined the subcellular distribution of the fusion proteins expressed from these plasmids using confocal microscopy of intact cells and Western blot analysis. DNA sequences coding for amino acids 1-139 and 1-136 of PGHS-1 and PGHS-2, respectively, which include the signal peptides, epidermal growth factor homology domains, glycosylation sites, and the putative membrane-binding helices of these two isozymes, were required for targeting the PGHS-green fluorescent protein fusion proteins to the endoplasmic reticulum and nuclear membranes when expressed in NIH 3T3 cells. Chimeric proteins that did not contain the putative membrane binding domains are targeted to the endoplasmic reticulum, but are not associated with membrane structures, and are present only in soluble cell fractions. These are the first experiments to directly confirm that the amphipathic helices present near the amino terminus of the PGHS-1 and PGHS-2 isozymes act as membrane anchors.
Li
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Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Smith
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Grabski
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DeWitt
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DL
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D017931:DNA Primers; D003902:Detergents; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism
D017931:DNA Primers; D003902:Detergents; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011451:Prostaglandin-Endoperoxide Synthases