Analysis of nuclear pore protein p62 glycosylation.
Glycoprotein components of the nuclear pore are essential for nuclear transport and are modified by both glycosylation and phosphorylation. The function and control of these post-translational modifications are poorly understood. Glycosylation of the major rat nuclear pore glycoprotein, p62, was examined in vitro using recombinant p62 as a substrate. Rat p62 was expressed in Escherichia coli and purified to near homogeneity. Kinetic analysis using a partially purified mammalian transferase suggests that the recombinant protein is an excellent substrate (Km = 0.30 microM) for the transfer of GlcNAc from UDP-GlcNAc (Km = 1.8 microM). Localization of the sites of O-linked GlcNAc glycosylation of rat p62 was performed by a combination of deletion analysis of in vitro translation products and by immunoprecipitation of [14C]GlcNAc-labeled proteolytic fragments. The amino terminus of rat p62 is poorly glycosylated with no O-linked GlcNAc sites between Lys22 and Lys97; the carboxyl terminus has one known glycosylation site at Ser471. The majority of the glycosylation sites in rat p62 are likely to occur on the six clustered Ser residues in the central Ser/Thr-rich region from Ser270 to Thr294. A synthetic peptide derived from this region is a good substrate for O-GlcNAc addition (Km = 30 microM) and a potent competitive inhibitor of p62 glycosylation (Ki = 15 microM). It is proposed that this Ser/Thr-rich domain functions as a linker region between the amino-terminal beta-pleated sheet and the carboxyl terminal alpha-helical domains. O-Glycosylation and phosphorylation of this linker region could provide a dynamic means of altering the conformation of p62 during nuclear pore assembly and disassembly.
Laboratory of Biochemistry and Metabolism, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
W A
WA
Lubas
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M
Smith
C M
CM
Starr
J A
JA
Hanover
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D006031:Glycosylation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018272:Porins / Q000737:chemistry* / Q000235:genetics; D051381:Rats
D018076:DNA, Complementary; D018272:Porins