Subcellular localization and topology of �(1?4)galactosyltransferase that elongates �(1?4)galactan side chains in rhamnogalacturonan�I in potato

Geshi, Naomi; Jørgensen, Bodil; Ulvskov, Peter

doi:10.1007/s00425-003-1168-3

Cited by 21 publications

(14 citation statements)

References 36 publications

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“…The subcellular localization of GALS1 was investigated by heterologous expression of a yellow fluorescent protein (YFP) fusion in Nicotiana benthamiana and confocal laser scanning microscopy. This analysis showed that GALS1 was targeted to Golgi vesicles, consistent with a role in pectin biosynthesis and the reported location of b-1,4-galactan synthase activity (Geshi et al, 2004) (Figure 4). GALS2 and GALS3 have previously been identified as putative Golgi proteins in a proteomic study of purified Golgi vesicles from Arabidopsis cell cultures (Parsons et al, 2012).…”

Section: Gals1 Is Located In the Golgi Apparatussupporting

confidence: 83%

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase

et al. 2012

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b-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include a-1,5-arabinans, b-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but b-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased b-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher b-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto b-1,4-galactopentaose. GALS1 specifically formed b-1,4-galactosyl linkages and could add successive b-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as b-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.

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Section: Gals1 Is Located In the Golgi Apparatussupporting

confidence: 83%

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase

et al. 2012

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“…It has been known for a long time that noncellulosic polysaccharides are synthesized in the Golgi apparatus (Ray et al, 1969), and enzymes involved in the synthesis of the backbone of noncellulosic b-linked CW polysaccharides (e.g., the mannan synthases) have been shown to be Golgi localized, multimembranespanning enzymes (Dhugga et al, 2004;Liepman et al, 2005). Evidence pointing toward pectin also being synthesized in the Golgi has been reviewed by Staehelin and Moore (1995), and it has since then been substantiated by biochemical evidence that three enzymes involved in pectin biosynthesis are bound to the Golgi membrane with the active site facing the lumen (Goubet and Mohnen, 1999;Sterling et al, 2001;Geshi et al, 2004). It is therefore very likely that RG-II is also assembled in the Golgi vesicles, which is supported by the accumulation of RGXT1-EGFP and RGXT2-EGFP fusion proteins in an intracellular compartment that disintegrates upon treatment with BFA.…”

Section: Protein Structure and Subcellular Localizationmentioning

confidence: 99%

“…Different epitopes present in pectin have been detected in various compartments of the Golgi apparatus (Staehelin and Moore, 1995;Willats et al, 2001), and enzymes synthesizing pectin have been detected in Golgiderived vesicles (Goubet and Mohnen, 1999;Sterling et al, 2001;Geshi et al, 2004), indicating that pectin is indeed synthesized in the Golgi apparatus. Glycosyltransferases (GTs) located in the Golgi apparatus typically have a type II membrane protein structure consisting of a single N-terminal transmembrane domain (TMD), followed by a stem-like region of variable length and the C-terminal catalytic domain facing the lumen of the Golgi apparatus (Breton et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Arabidopsis thaliana RGXT1 and RGXT2 Encode Golgi-Localized (1,3)-α-d-Xylosyltransferases Involved in the Synthesis of Pectic Rhamnogalacturonan-II

Egelund¹,

Petersen²,

Motawia³

et al. 2006

The Plant Cell

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126

145

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Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-a-D-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative of a type II membrane protein structure. Soluble secreted forms of the corresponding proteins expressed in insect cells showed xylosyltransferase activity, transferring D-xylose from UDPa-D-xylose to L-fucose. The disaccharide product was hydrolyzed by a-xylosidase, whereas no reaction was catalyzed by b-xylosidase. Furthermore, the regio-and stereochemistry of the methyl xylosyl-fucoside was determined by nuclear magnetic resonance to be an a-(1,3) linkage, demonstrating the isolated glycosyltransferases to be (1,3)-a-D-xylosyltransferases. This particular linkage is only known in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1-and RGXT2-enhanced green fluorescent protein constructs in Arabidopsis revealed that both fusion proteins were targeted to a Brefeldin A-sensitive compartment and also colocalized with the Golgi marker dye BODIPY TR ceramide, consistent with targeting to the Golgi apparatus. Taken together, these results suggest that RGXT1 and RGXT2 encode Golgi-localized (1,3)-a-D-xylosyltransferases involved in the biosynthesis of pectic rhamnogalacturonan-II.

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“…More recent topology studies of such glycosyltransferases include the protection of enzyme activities from protease treatment by the Golgi membranes, for the -1, 4-galacturonosyltransferase involved in the biosynthesis of homogalacturonan (Sterling et al 2001), and the -1, 4-galactosyltransferase involved in the synthesis of -1, 4-galactan side chains in rhamnogalacturonan I (Geshi et al 2004). On the other hand, the maize mixed linkage (1-3), (1-4)--D-glucan synthase was found to be sensitive to pretreatments of Golgi membranes with proteinase K (Urbanowicz et al 2004).…”

Section: Introductionmentioning

confidence: 99%

AtCSLD2 is an integral Golgi membrane protein with its N-terminus facing the cytosol

Zeng

Keegstra

2008

Planta

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Cellulose synthase-like proteins in the D family share high levels of sequence identity with the cellulose synthase proteins and also contain the processive -glycosyltransferase motifs conserved among all members of the cellulose synthase superfamily. Consequently, it has been hypothesized that members of the D family function as either cellulose synthases or glycan synthases involved in the formation of matrix polysaccharides. As a prelude to understanding the function of proteins in the D family, we sought to determine where they are located in the cell. A polyclonal antibody against a peptide located at the N-terminus of the Arabidopsis D2 cellulose synthase-like protein was generated and puriWed. After resolving Golgi vesicles from plasma membranes using endomembrane puriWcation techniques including two-phase partitioning and sucrose density gradient centrifugation, we used antibodies against known proteins and marker enzyme assays to characterize the various membrane preparations. The Arabidopsis cellulose synthase-like D2 protein was found mostly in a fraction that was enriched with Golgi membranes. In addition, versions of the Arabidopsis cellulose synthase-like D2 proteins tagged with a green Xuorescent protein was observed to colocalize with a DsRed-tagged Golgi marker protein, the rat alpha-2,6-sialyltransferase. Therefore, we postulate that the majority of Arabidopsis cellulose synthase-like D proteins, under our experimental conditions, are likely located at the Golgi membranes. Furthermore, protease digestion of Golgi-rich vesicles revealed almost complete loss of reaction with the antibodies, even without detergent treatment of the Golgi vesicles. Therefore, the N-terminus of the Arabidopsis cellulose synthase-like D2 protein likely faces the cytosol. Combining this observation with the transmembrane domain predictions, we postulate that the large hydrophilic domain of this protein also faces the cytosol.

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Subcellular localization and topology of �(1?4)galactosyltransferase that elongates �(1?4)galactan side chains in rhamnogalacturonan�I in potato

Cited by 21 publications

References 36 publications

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase

Arabidopsis thaliana RGXT1 and RGXT2 Encode Golgi-Localized (1,3)-α-d-Xylosyltransferases Involved in the Synthesis of Pectic Rhamnogalacturonan-II

AtCSLD2 is an integral Golgi membrane protein with its N-terminus facing the cytosol

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