Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine.

Abstract

Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized. As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc). Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs. Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays. Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues. Most of these appear to be bound to proteins via a hydroxyl group. This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs.

O-glycosylation of protein subpopulations in alcohol-extracted rice proteins

“…O-glycosylation of the precursor to sporamin, a vacuolar protein of the sweet potato, has also been shown to occur in the Golgi apparatus (Matsuoka et al, 1995). Several novel oligosaccharides with terminal GlcNAc modification, the majority of which are O-linked, have been identified on nuclear pore proteins from suspension-cultured tobacco cells (Heese-Peck and Raikhel, 1998;Heese-Peck et al, 1995). The latter reports have been erroneously referenced as documenting O-GlcNAc modification of proteins or the presence of a 'polyGlcNAc' chain in plants (Meier, 2001;Thornton et al, 1999).…”

“…Terminal GlcNAc of membrane-bound proteins was detected by labeling with [ 3 H] galactose (Heese-Peck et al, 1995;Kamemura et al, 2002;Roquemore et al, 1994). Following labeling, the membrane was sprayed with EN 3 HANCE (PerkinElmer Life Sciences, Inc.; Boston, MA), air dried in a fume hood and exposed to pre-flashed (Laskey and Mills, 1975) BioMax XAR film (Eastman Kodak; Rochester, NY) at À80 1C.…”

The plant nuclear envelope

“…Early candidates for plant nucleoporins included a 100-kDa protein in the nuclear matrix of carrot suspension cells, which was recognized by an antibody against the yeast nucleoporin Nsp1p (Scofield et al 1992). A number of proteins associated with the tobacco NPC were shown to be modified by N-acetylglucosamine (GlcNAc), a glycosylation frequently found in animal nucleoporins (Heese-Peck et al 1995).…”

Glycobiology and Immunology

“…O-GlcNAc had two novel aspects: First, as mentioned, it had a nucleocytoplasmic distribution, whereas "traditional" glycoproteins were localized to the cell surface and topologically equivalent intracellular compartments, such as the lumens of the endoplasmic reticulum and Golgi apparatus [78]. Second, with the possible exception of plant nuclear pore proteins [79], O-GlcNAc is not elongated into more complex structures but rather remains as a single monosaccharide on the peptide backbone. As such, the biosynthetic "machinery" for O-GlcNAc is extremely simple-with an important caveat mentioned below regarding the dynamic nature of this modification-compared to other types of glycosylation.…”