Glucosylation Activity and Complex Formation of Two Classes of Reversibly Glycosylated Polypeptides

Abstract

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch. We have isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function of RGPs. Sequence comparisons showed the existence of two classes of RGP proteins, designated RGP1 and RGP2. Glucosylation activity of RGP1 and RGP2 from wheat and rice was studied. After separate expression of Rgp1 and Rgp2 in Escherichia coli or yeast (Saccharomyces cerevisiae), only RGP1 showed self-glucosylation. In Superose 12 fractions from wheat endosperm extract, a polypeptide with a molecular mass of about 40 kD is glucosylated by UDP-glucose. Transgenic tobacco (Nicotiana tabacum) plants, overexpressing either wheat Rgp1 or Rgp2, were generated. Subsequent glucosylation assays revealed that in RGP1-containing tobacco extracts as well as in RGP2-containing tobacco extracts UDP-glucose is incorporated, indicating that an RGP2-containing complex is active. Gel filtration experiments with wheat endosperm extracts and extracts from transgenic tobacco plants, overexpressing either wheat Rgp1 or Rgp2, showed the presence of RGP1 and RGP2 in high-molecular mass complexes. Yeast two-hybrid studies indicated that RGP1 and RGP2 form homo-and heterodimers. Screening of a cDNA library using the yeast two-hybrid system and purification of the complex by an antibody affinity column did not reveal the presence of other proteins in the RGP complexes. Taken together, these results suggest the presence of active RGP1 and RGP2 homo-and heteromultimers in wheat endosperm.Reversibly glycosylated polypeptides (RGPs) are thought to be involved in polysaccharide metabolism. Polysaccharides are the main components of the plant cell wall. In dicots, 20% of the primary cell wall consists of the polysaccharide xyloglucan, whereas in monocots, this hemicellulose makes up 2% of the primary cell wall (Darvill et al., 1980). Xyloglucan consists of a ␤-1,4-glucan backbone with xylosyl and xylosyl-galactosyl-fucosyl side chains (Hayashi, 1989). The transferases responsible for the addition of these side chains are localized to the Golgi apparatus (Brummell et al., 1990;Driouich et al., 1993;Staehelin and Moore, 1995). RGP1 has been implicated in xyloglucan biosynthesis in pea (Pisum sativum; Dhugga et al., 1997). This protein, a 40-kD doublet, which could be glycosylated with radiolabeled UDP-Glc in the presence of Mn 2ϩ or Mg 2ϩ , is associated with Golgi membranes as shown by density gradient centrifugation (Dhugga et al., 1991). Immunolocalization experiments showed RGP1 to be present in transGolgi dictyosomal cisternae (Dhugga et al., 1997). An Arabidopsis homologue was found to be mostly soluble, but also to be associated with membranes (Delgado et al., 1998). Glycosylation of the pea protein with UDP-Glc, UDP-Xyl, and UDP-Gal in a ratio similar to that of the typical sugar composition of xyloglucan (UDP-Glc:UDP-Xyl:UDP-Gal ϭ 10...

Reduced levels of class 1 reversibly glycosylated polypeptide increase intercellular transport via plasmodesmata

“…Given their Golgi localization and glycosylation activity, RGPs have been implicated in polysaccharide biosynthesis, particularly cell wall and starch synthesis. 15,23 However there is little direct evidence to support these hypotheses. The strongest evidence for a role in cell wall synthesis derives from studies of pollen development in Arabidopsis.…”

Combined Proteomic and Cytological Analysis of Ca2+-Calmodulin Regulation inPicea meyeriPollen Tube Growth

“…an esterase/lipase [spot 57]). Reversibly glycosylated polypeptides (spots 21, 42, 55, and 67) have been implicated in polysaccharide biosynthesis and may function in cell wall construction and starch synthesis in plants (Langeveld et al, 2002).…”

Purification and Characterization of UDP-Arabinopyranose Mutase fromChlamydomonas reinhardtii

“…Individual expression of OsUAM1 and UAM2 in E. coli results in homo-hexamers. 25) These suggests that UAM forms homo-and hetero-complexes. In the presents study, the molecular weight of CrUAM was determined to be 39 kDa by SDS-PAGE and about 700 kDa by size-exclusion chromatography (Figs.…”

“…Chlamydomonas reinhardtii wild-type strain cc125+ (mt+) was grown in tris-acetate-phosphate (TAP) medium 22) at 25 C under a 12 h dark/12 h light (25 mmol photon m À2 s À1 ) cycle. Cells at late logarithmic phase (5{6 Â 10 6 cells mL À1 ) were harvested by centrifugation (2;250 Â g, 10 min) and stored at À80 C.…”

“…Overexpression of CrUAM in E. coli was performed as described previously. 18) E. coli cells carrying the pET 24a/CrUAM were grown at 37 C and further cultured at 25 C for 3 h in the presence of 0.1 mM isopropylthio -D-galactopyranoside. The cells were harvested and sonicated to solubilize the cytosol proteins.…”