CHARACTERIZATION OF NUCLEAR-PORE PROTEIN P62 PRODUCED USING BACULOVIRUS

Nuclear pore glycoproteins are essential components of the nuclear imp ort apparatus in eukaryotes. In vertebrates, the most abundant of thes e glycoproteins is a molecule called p62, Like other O-linked N-acetyl glucosamine glycoproteins, p62 is normally modified in the cytoplasm a nd cannot be overexpressed and conveniently collected in a secreted fo rm. We devised an efficient scheme for expression and purification of recombinant p62 from Sf9 cells that may have general applicability for this class of glycoproteins. The purified rat p62 bound to wheat germ agglutinin, consistent with modification by O-linked N-acetylglucosam ine. Carbohydrate analysis, in conjunction with amino acid analysis, r evealed that baculovirus-expressed rat p62 contains 5-6 mol of N-acety lglucosamine/mol of p62, As observed by circular dichroism, purified p 62 expressed in the baculovirus system or in Escherichia coli share es sentially the same secondary structure, Purified glycosylated rat p62 will be critical in determining the role of N-acetylglucosamine in bot h nuclear transport and assembly of the nuclear pore complex. (C) 1995 Academic Press, Inc.