Solubilization and properties of GDP-fucose: Xyloglucan 1,2-α-l-fucosyltransferase from pea epicotyl membranes

Hanna, Rami; Brummell, David A.; Camirand, Anne; Hensel, Andreas; Russell, Elizabeth F.; Maclachlan, Gordon

doi:10.1016/0003-9861(91)90584-6

Cited by 38 publications

(16 citation statements)

References 32 publications

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“…To answer some of these questions, we analyzed the orientation of the branching enzyme xyloglucan ␣,1-2 fucosyltransferase (XG-FucTase) in Golgi vesicles from pea (Pisum sativum) stems. This enzyme uses GDP-Fuc as a substrate and is responsible for the addition of the terminal Fuc onto xyloglucan (Camirand et al, 1987;Farkas and Maclachlan, 1988;Hanna et al, 1991). Our results indicate that XG-FucTase has its active site in the lumen of the Golgi cisternae.…”

mentioning

confidence: 73%

GDP-Fucose Uptake into the Golgi Apparatus during Xyloglucan Biosynthesis Requires the Activity of a Transporter-Like Protein Other Than the UDP-Glucose Transporter

2000

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The molecular mechanisms regulating hemicelluloses and pectin biosynthesis are poorly understood. An important question in this regard is how glycosyltransferases are oriented in the Golgi cisternae, and how nucleotide sugars are made available for the synthesis of the polymers. Here we show that the branching enzyme xyloglucan ␣,1-2 fucosyltransferase (XG-FucTase) from growing pea (Pisum sativum) epicotyls was latent and protected against proteolytic inactivation on intact, right-side-in pea stem Golgi vesicles. Moreover, much of the XG-FucTase activity was membrane associated. These data indicate that XG-FucTase is a membrane-bound luminal enzyme. GDP-Fuc uptake studies demonstrated that GDP-Fuc was taken up into Golgi vesicles in a protein-mediated process, and that this uptake was not competed by UDP-Glc, suggesting that a specific GDP-Fuc transporter is involved in xyloglucan biosynthesis. Once in the lumen, Fuc was transferred onto endogenous acceptors, including xyloglucan. GDPase activity was detected in the lumen of the vesicles, suggesting than the GDP produced upon transfer of Fuc was hydrolyzed to GMP and inorganic phosphate. We suggest than the GDP-Fuc transporter and GDPase may be regulators of xyloglucan fucosylation in the Golgi apparatus from pea epicotyls.

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mentioning

confidence: 73%

GDP-Fucose Uptake into the Golgi Apparatus during Xyloglucan Biosynthesis Requires the Activity of a Transporter-Like Protein Other Than the UDP-Glucose Transporter

2000

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“…Vesicles enriched in Golgi complex membranes were subjected to proteinase K treatment, then assayed for XyG FUTase activity and resolved on SDS-PAGE for immunoblotting. XyG FUTase activity is a latent Golgi activity because permeabilization of the Golgi membranes by detergent is required for measurement of its activity (Hanna et al 1991;WulV et al 2000). Therefore, we used the XyG FUTase activity as a marker for the inside of the Golgi complex membrane.…”

Section: Atcsld2-gfp Colocalized With St-dsred At Golgi Vesicles Whenmentioning

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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“…The assay for the activity of the Golgi marker enzyme xyloglucan 1,2-a-~-fucosyltransferase was carried out essentially as described previously (Hanna et al, 1991). The standard assay system consisted of 200 pL of a buffer containing 25 mM Pipes-KOH, pH 6.5, 2 mM MgCI,, 0.5 mg mL-' purified soluble tamarind xyloglucan, 0.5% Triton X-114, and 0.7 mM GDP-[3H]Fuc (740 Bq/fraction), and 100 pL of a protein sample solubilized with Triton X-114 (final lo/o) from fractions of the SUC density gradients that were prepared in the presence of EDTA.…”

Section: Golgi Marker Enzyme Assaymentioning

confidence: 99%

“…Enzyme activity for xyloglucan 1,2-a-L-fucosyltransferase was also used as a marker for the Golgi (Hanna et al, 1991). In step Sue density gradients (Fig.…”

Section: Atelp Is An Integral Membrane Protein Found In a Novel Compamentioning

confidence: 99%

Cloning and Subcellular Location of an Arabidopsis Receptor-Like Protein That Shares Common Features with Protein-Sorting Receptors of Eukaryotic Cells

1997

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Many receptors involved in clathrin-mediated protein transport through the endocytic and secretory pathways of yeast and animal cells share common features. They are all type I integral membrane proteins containing cysteine-rich lumenal domains and cytoplasmic tails with tyrosine-containing sorting signals. The cysteine-rich domains are thought to be involved in ligand binding, whereas the cytoplasmic tyrosine motifs interact with clathrin-associated adaptor proteins during protein sorting along these pathways. In addition, tyrosine-containing signals are required for the retention and recycling of some of these membrane proteins to the trans-Golgi network. Here we report the characterization of an approximately 80-kD epidermal growth factor receptor-like type I integral membrane protein containing all of these functional motifs from Arabidopsis thaliana (called AtELP for A. thaliana Epidermal growth factor receptor-Like Protein). Biochemical analysis indicates that AtELP is a membrane protein found at high levels in the roots of both monocots and dicots. Subcellular fractionation studies indicate that the AtELP protein is present in two membrane fractions corresponding to a novel, undefined compartment and a fraction enriched in vesicles containing clathrin and its associated adaptor proteins. AtELP may therefore serve as a marker for compartments involved in intracellular protein trafficking in the plant cell.

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Solubilization and properties of GDP-fucose: Xyloglucan 1,2-α-l-fucosyltransferase from pea epicotyl membranes

Cited by 38 publications

References 32 publications

GDP-Fucose Uptake into the Golgi Apparatus during Xyloglucan Biosynthesis Requires the Activity of a Transporter-Like Protein Other Than the UDP-Glucose Transporter

GDP-Fucose Uptake into the Golgi Apparatus during Xyloglucan Biosynthesis Requires the Activity of a Transporter-Like Protein Other Than the UDP-Glucose Transporter

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

Cloning and Subcellular Location of an Arabidopsis Receptor-Like Protein That Shares Common Features with Protein-Sorting Receptors of Eukaryotic Cells

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