Structural requirements for Arabidopsisβ1,2‐xylosyltransferase activity and targeting to the Golgi

Abstract

SummaryCharacterization of a b1,2-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.

Cell biology of the plant Golgi apparatus

“…It was shown that the signal anchor sequence of a rat sialyl transferase (52 amino acids of the transmembrane domain and the cytoplasmic tail) and the equivalent sequence from a human galactosyl transferase was sufficient to target GFP to the Golgi stacks in tobacco leaf cells and BY2 suspension culture cells ( Boevink et al, 1998;Saint-Jore et al, 2002). Subsequently the equivalent sequences from recently cloned plant glycosyl transferases gave the same result ( Essl et al, 1999;Dirnberger et al, 2002;Pagny et al, 2003). These results suggest that the mechanisms of targeting and retention of glycosyl transferases may be conserved across kingdoms.…”

N-Glycans of Phaeodactylum tricornutum Diatom and Functional Characterization of Its N-Acetylglucosaminyltransferase I Enzyme

“…A putative type II xylosyltransferase is also predicted in the genome. This sequence shares 24% of identity with the luminal part of A. thaliana ␤(1,2)-xylosyltransferase involved in the transfer of a ␤(1,2)-xylose residue onto the ␤-Man of the N-glycan core (67,68). Nevertheless, in the absence of reported motifs specific for ␤(1,2)-xylosyltransferase activity, the involvement of this putative transferase in P. tricornutum N-glycan pathway remains highly hypothetical.…”

An antibody produced in tobacco expressing a hybrid β-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

“…Using immunoelectronmicroscopic detection of a GFP that was N-terminally tagged with the 36 amino acid residues of the Arabidopsis XylT, the hybrid fluorescent protein has been shown to be concentrated mainly in the medial Golgi (17). Because our xylGalT comprises as much as 53 amino acids of the N terminus of the same xylosyltransferase (i.e., the region containing the cytoplasmic tail and the transmembrane domain), we suggest that this hybrid enzyme is also likely to be located in the medial Golgi.…”

“…A number of studies using hybrid glycosyltransferases have established that the cytoplasmic tail, transmembrane domains, and stem regions (i.e., the CTS domains) of several of these enzymes, including those of human GalT and plant core ␤-1,2-xylosyltransferase (XylT), play a decisive role in their sub-Golgi distribution (11,16,17). In the current study, we examined whether altering the localization of human GalT in plant cells by swapping its natural CTS domain with one from Arabidopsis thaliana XylT, could be used to modify N-glycosylation in plants and to reduce fucosylation and xylosylation of the chitobiose core.…”