Localization of prostaglandin endoperoxide synthase-1 to the endoplasmic reticulum and nuclear envelope is independent of its C-terminal tetrapeptide-PTEL.
Prostaglandin endoperoxide H (PGH) synthases 1 and 2 are both membrane-associated proteins localized to the endoplasmic reticulum (ER) and nuclear envelope. The carboxyl terminal tetrapeptides of PGH synthases 1 and 2 are of the form -P/STEL. These sequences are similar to the -KDEL retention signal sequence characteristic of many proteins localized to the ER. To determine if the -PTEL sequence (residues 597-600) functions as an ER retention signal for ovine PGH synthase-1, we prepared and analyzed five mutants (L600N, L600R, L600V, E599Q, and delta 597), all having modifications that would be expected to alter the subcellular location of PGH synthase-1 if the -PTEL sequence were involved in ER targeting. Native ovine PGH synthase-1 and each of the five mutants were subcloned into the pSVT7 expression vector and were expressed transiently in cos-1 cells. The L600N, L600R, E599Q, and delta 597 mutants retained both cyclooxygenase and peroxidase activities. Moreover, when subjected to immunocytofluorescent staining, cos-1 cells expressing native and mutant enzymes showed similar patterns of fluorescence corresponding to ER and nuclear envelope localization. Finally, culture media bathing cos-1 cells transfected with native or mutant PGH synthases were tested for secreted PGH synthase-1 protein by Western blotting, but no PGH synthase-1 was detected in any of the culture media. Our results demonstrate that mutations in the C-terminal sequence-PTEL do not change the subcellular location of ovine PGH synthase-1. Thus, targeting of PGH synthase-1 to the ER can occur independent of its -PTEL sequence.
Regier
M K
MK
Department of Biochemistry, Michigan State University, East Lansing 48824.
Otto
J C
JC
DeWitt
D L
DL
Smith
W L
WL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009685:Nuclear Envelope / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry*; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis* / Q000737:chemistry*; D017385:Sequence Homology; D012756:Sheep; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D010446:Peptide Fragments; D011451:Prostaglandin-Endoperoxide Synthases