Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway.
Using a monoclonal antibody (mAb 414), we previously identified a protein of 62 kDa (p62) that was localized to the nuclear pore complex by immunoelectron microscopy. We also showed that p62 binds specifically to wheat germ agglutinin. Therefore, we proposed that this nuclear pore complex protein might be a member of a recently characterized family of glycoproteins that are labeled by in vitro galactosylation of rat liver nuclei and contain O-linked monosaccharidic GlcNAc residues. In support of this, we now show that incubation with N-acetylglucosaminidase reduces the molecular mass of p62 by approximately 3 kDa because of the removal of terminal GlcNAc residues. Moreover, p62 can be galactosylated in vitro by using UDP-[3H]galactose and galactosyltransferase. We also show that most of the GlcNAc residues are added within 5 min of synthesis, when p62 is soluble and cytosolic. Thus, the addition of GlcNAc is carried out in the cytoplasm and is clearly distinct from the N- and O-linked glycosylation pathways of the endoplasmic reticulum and Golgi complex. Using another mAb with a broad specificity for nuclear GlcNAc-containing proteins, we show by immunofluorescence and protein blotting of subnuclear fractions that some of these proteins are in the interior of the nucleus, and others are most likely located in the pore complex.
Davis
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LI
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021.
Blobel
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G
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000096:biosynthesis*; D006026:Glycoside Hydrolases; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000378:metabolism*; D010449:Peptide Mapping; D051381:Rats; D011915:Rats, Inbred BUF; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D006023:Glycoproteins; D014531:Uridine Diphosphate Galactose; D006026:Glycoside Hydrolases