Glycosylation of nuclear pore protein p62. Reticulocyte lysate catalyzes O-linked N-acetylglucosamine addition in vitro.
The addition of O-linked N-acetylglucosamine (GlcNAc) to the major nuclear pore complex glycoprotein p62 was examined. Expression of the rat p62 cDNA in transfected monkey cells was detected using a rat p62-specific antipeptide antiserum and two previously described nuclear pore-specific monoclonal antibodies which require O-linked GlcNAc for binding. Although the p62 cDNA was predicted to encode a 54-kDa polypeptide, the product expressed in monkey cells migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two species of 62 and 59-kDa. Cell-free translation of the p62 in vitro transcript yielded a 59-kDa polypeptide using wheat germ extract and a 62-kDa product using a commercially available rabbit reticulocyte lysate. Several lines of evidence indicated that the 62-kDa rabbit reticulocyte lysate translation product was modified by O-linked N-acetylglucosamine; the protein bound specifically to a wheat germ agglutinin affinity column and was converted to 59 kDa when treated with jack bean beta-acetylglucosaminidase. The 59-kDa unglycosylated wheat germ translation product was converted to the 62-kDa glycosylated form upon incubation with reticulocyte lysate demonstrating that O-linked GlcNAc can be added to p62 post-translationally.
Starr
C M
CM
Laboratory of Biochemistry and Metabolism, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Hanover
J A
JA
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005944:Glucosamine / Q000031:analogs & derivatives*; D006031:Glycosylation; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D028861:Nuclear Pore Complex Proteins; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism*; D014162:Transfection
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; C049312:nuclear pore protein p62; D004247:DNA; D005944:Glucosamine; D000117:Acetylglucosamine