Prostaglandin H synthase-1: evaluation of C-terminus function.
The first committed step in prostaglandin biosynthesis is catalyzed by prostaglandin H synthase, an enzyme localized to the endoplasmic reticulum (ER) membrane in a variety of cells. Several types of C-terminal region peptide sequence motifs have been found to lead to ER retention of other proteins. We have tested the potential role for such signals in the ER localization and catalytic activity of human isoform-1 of the synthase (PGHS-1). PGHS-1 mutants with alterations in the C-terminus designed to disrupt potential retention signals were expressed in transfected COS-1 cells. The mutations included: substitution of valine for the ultimate leucine residue (position 600) to disrupt a KDEL-type signal, substitution of a neutral glutamine for arginine at position 595 to disrupt signals based on positive charge, and deletion of the last six residues, to remove all of the wild-type extreme C-terminus. The subcellular localization of each recombinant PGHS-1 was assessed by differential centrifugation and by immunofluorescence microscopy. None of the mutations led to a significant change in the distribution of PGHS-1 between microsomes and other cellular fractions. Immunostaining of wild-type PGHS-1 and all of the mutants colocalized with that of protein disulfide isomerase, an ER marker protein. However, mutation of the terminal leucine or deletion of the last six residues did lead to loss of cyclooxygenase activity. Mutation of the terminal leucine also altered the pattern of fragments produced by limited proteolysis, indicating that this mutation led to changes in the polypeptide folding which might account for the loss of activity. The results indicate that the extreme C-terminal region is important to the functional integrity of PGHS-1, but it is not an essential part of the intracellular targeting mechanism.
Ren
Y
Y
Department of Internal Medicine, University of Texas Health Science Center at Houston 77030.
Loose-Mitchell
D S
DS
Kulmacz
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D011451:Prostaglandin-Endoperoxide Synthases