Structural requirements for Arabidopsis beta1,2-xylosyltransferase activity and targeting to the Golgi.
Characterization of a beta1,2-xylosyltransferase from Arabidopsis thaliana (AtXylT) was carried out by expression in Sf9 insect cells using a baculovirus vector system. Serial deletions at both the N- and C-terminal ends proved that integrity of a large domain located between amino acid 31 and the C-terminal lumenal region is required for AtXylT activity expression. The influence of N-glycosylation on AtXylT activity has been evaluated using either tunicamycin or mutagenesis of potential N-glycosylation sites. AtXylT is glycosylated on two of its three potential N-glycosylation sites (Asn51, Asn301, Asn478) and the occupancy of at least one of these two sites (Asn51 and Asn301) is necessary for AtXylT stability and activity. Contribution of the N-terminal part of AtXylT in targeting and intracellular distribution of this protein was studied by expression of variably truncated, GFP-tagged AtXylT forms in tobacco cells using confocal and electron microscopy. These studies have shown that the transmembrane domain of AtXylT and its short flanking amino acid sequences are sufficient to specifically localize a reporter protein to the medial Golgi cisternae in tobacco cells. This study is the first detailed characterization of a plant glycosyltransferase at the molecular level.
Pagny
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S
CNRS UMR 6037, IFRMP 23, Université de Rouen, UFR des Sciences, Bâtiment extension Biologie 76821, Mont St Aignan Cedex, France.
Bouissonnie
F
F
Sarkar
M
M
Follet-Gueye
M L
ML
Driouich
A
A
Schachter
H
H
Faye
L
L
Gomord
V
V
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000648:ultrastructure; D001216:Asparagine; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010430:Pentosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D001216:Asparagine; D010430:Pentosyltransferases; C408586:beta 1,2-xylosyltransferase