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First published online September 3, 2008; 10.1104/pp.108.127027 Plant Physiology 148:1354-1367 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Comparative Analyses of Arabidopsis complex glycan1 Mutants and Genetic Interaction with staurosporin and temperature sensitive3a1,[W],[OA]Institut für Botanik, Westfälische Wilhelms-Universität Münster, 48149 Munster, Germany (J.F., H.K.-S., S.R., A.v.S.); and Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, and Molecular and Environmental Plant Science Program, Texas A&M University, College Station, Texas 77843–2133 (H.K.)
We compare three Arabidopsis (Arabidopsis thaliana) complex glycan1 (cgl1) alleles and report on genetic interaction with staurosporin and temperature sensitive3a (stt3a). STT3a encodes a subunit of oligosaccharyltransferase that affects efficiency of N-glycan transfer to nascent secretory proteins in the endoplasmic reticulum; cgl1 mutants lack N-acetyl-glucosaminyltransferase I activity and are unable to form complex N-glycans in the Golgi apparatus. By studying CGL1-green fluorescent protein fusions in transient assays, we show that the extra N-glycosylation site created by a point mutation in cgl1 C5 is used in planta and interferes with folding of full-length membrane-anchored polypeptides in the endoplasmic reticulum. Tunicamycin treatment or expression in the stt3a-2 mutant relieved the folding block, and migration to Golgi stacks resumed. Complementation tests with C5-green fluorescent protein and other N-glycosylation variants of CGL1 demonstrated that suppression of aberrant N-glycosylation restores activity. Interestingly, CGL1 seems to be functional also as nonglycosylated enzyme. Two other cgl1 alleles showed splicing defects of their transcripts. In cgl1 C6, a point mutation affects the 3' splice site of intron 14, resulting in frame shifts; in cgl1-T, intron 11 fails to splice due to insertion of a T-DNA copy. Introgression of stt3a-2 did not restore complex glycan formation in cgl1 C6 or cgl1-T but suppressed the N-acetyl-glucosaminyltransferase I defect in cgl1 C5. Root growth assays revealed synergistic effects in double mutants cgl1 C6 stt3a-2 and cgl1-T stt3a-2 only. Besides demonstrating the conditional nature of cgl1 C5 in planta, our observations with loss-of-function alleles cgl1 C6 and cgl1-T in the stt3a-2 underglycosylation background prove that correct N-glycosylation is important for normal root growth and morphology in Arabidopsis.
N-Glycosylation of secreted proteins is a vital function in all eukaryotic cells. N-Glycans attached to glycoproteins support proper folding in the endoplasmic reticulum (ER) lumen (a prerequisite for vesicle-mediated protein export to the cis-Golgi), increase hydration at the protein surface, and confer enhanced stability against proteolytic degradation. During cotranslational import into the ER, most secretory proteins are glycosylated on Asn residues within the conserved motif [Asn-X(
 ). Inhibition of core N-glycosylation by the nucleoside antibioticum tunicamycin (for review, see Elbein, 1987) or mutations abrogating OST activity in the ER lumen activates the unfolded protein response (Koizumi et al., 1999; Martinez and Chrispeels, 2003; for review, see Patil and Walter, 2001; Lai et al., 2007) and ER-associated degradation (Bonifacino and Weissman, 1998; Plemper and Wolf, 1999; Parodi, 2000). While prolonged activation of the unfolded protein response results in lethality (Silberstein et al., 1995; Koizumi et al., 1999; Iwata and Koizumi, 2005; Hauptmann et al., 2006, and refs. therein), unfolded protein response and ER-associated degradation operate together as part of the quality-control surveillance in the secretory system of eukaryotes (Ellgaard et al., 1999; Hebert et al., 2005; Banerjee et al., 2007). Only when folding of single polypeptide chains and assembly of multisubunit protein complexes are completed are glycoproteins released and transported from the ER to the cis-Golgi via SNARE-mediated vesicle-budding and fusion events (Chatre et al., 2005; Moreau et al., 2007).
During passage through the various Golgi cisternae, surface-accessible N-glycans of plant glycoproteins (Faye et al., 1986
For cgl1 C5, Strasser et al. (2005)
Novel cgl1 C6 and cgl1-T Mutants Produce CGL1 Transcripts with Splicing Defects
We addressed the molecular basis for missing complex glycan formation in three available Arabidopsis cgl1 alleles. Mutants cgl1 C5 and C6 were originally identified in a screen of EMS-mutagenized M2 seedlings (von Schaewen et al., 1993 For cgl1 C6, we established that the G-to-A change at position 2,556 alters the 3' splice site of intron 14 (Fig. 1A ). Insertion mutant cgl1-T was identified in the SIGnAL-SALK database. Genomic PCR analyses confirmed that a single T-DNA resides within intron 11 (Supplemental Fig. S1A). Sequence analyses of both border fragments located the T-DNA insertion at position 1,792 without any base deletion.
In order to confirm the predicted molecular defects in cgl1 C6 and cgl1-T at the transcript level, we characterized the structures of mutant CGL1 transcripts by reverse transcription (RT)-PCR and sequence analyses. For cgl1 C6, sequencing the products amplified with Ex12/13_F and Ex18_R (Fig. 1B) revealed that intron 14 is aberrantly spliced by shifting the 3' splice site to the next G, introducing a translational frame shift (Fig. 1A). Larger PCR products found in cgl1 C6 were aberrant transcripts that retain intron 15, 16, and 17 (Fig. 1B; Supplemental Fig. S1C). For cgl1-T, transcripts were characterized using primers binding upstream or flanking the T-DNA insertion. RT-PCR with upstream primer pairs 5'_F and Ex3_R detected wild-type transcript levels; however, PCR using primer pairs Ex6_F and Ex12_R flanking the T-DNA insertion yielded no product for cgl1-T (Fig. 1B). This demonstrated that cgl1-T does not produce intact CGL1 transcripts. Since we could detect chimeric CGL1:T-DNA transcripts (Supplemental Fig. S1B), it appears that mature CGL1-T transcripts contain unspliced intron 11 with an additional 4.5 kb of T-DNA sequence. In both cgl1 C6 and cgl1-T, the mutations create premature stop codons a few codons downstream of the mutated sites (Fig. 1C).
Due to the D
In order to determine whether reduced frequency of N-glycosylation can specifically restore GnTI function of CGL1 C5 in planta, the viable stt3a-2 allele was introgressed into all three Arabidopsis cgl1 lines. STT3a encodes an isoform of OST subunit STT3, and the stt3a-2 mutation was previously shown to cause underglycosylation of glycoproteins (Koiwa et al., 2003 ). Both double mutants cgl1 C6 stt3a-2 and cgl1-T stt3a-2 failed to react with complex glycan antiserum on immunoblots ("nonstainers"; Fig. 2B). This, however, was not the case for the cgl1 C5 stt3a-2 double mutant lines. Compared with the wild type and the cgl1 single mutants, cgl1 C5 stt3a-2 displayed intermediate complex glycosylation patterns on immunoblots (Supplemental Fig. S2). It is noteworthy that the complex glycosylation pattern and intensity in cgl1 C5 stt3a-2 match exactly those of the stt3a parent (Fig. 2B). Thus, despite the D 144N change in a highly conserved GnTI motif (Wenderoth and von Schaewen, 2000; Strasser et al., 2005), CGL1 C5 polypeptides appear to retain sufficient activity to restore complex glycosylation in the stt3a mutant.
In order to test the N-glycosylation status of CGL1 variants, cDNA fragments encoding wild-type and mutated CGL1 variants were fused to GFP (Fig. 3A
) and expressed in Arabidopsis wild-type and stt3a-2 mutant protoplasts. The variants of CGL1 tested include C5, CGL1* (lacking native glycosylation site 351N), and C5* (lacking native glycosylation site 351N but containing aberrant glycosylation site 144N). Experiments were conducted in both the presence and absence of proteasome inhibitor MG-132 to prevent potential degradation of aberrant fusion proteins (Lee and Goldberg, 1998
Aberrant Glycosylation of CGL1 C5 Polypeptides Prevents Their Migration to Golgi Stacks
Since aberrant glycosylation of CGL1 C5 polypeptides may also affect Golgi targeting, we determined the subcellular location of fluorescent protein fusions using confocal laser-scanning microscopy. In transfected Arabidopsis protoplasts, CGL1-GFP and CGL1-OFP (for orange-shifted monomeric red fluorescent protein [mRFP]) both labeled Golgi stacks, as indicated by shape, size, and characteristic stop-and-go movements (Nebenführ et al., 1999 In the presence of tunicamycin, Col wild-type protoplasts transformed with either C5-GFP or C5*-GFP constructs developed a typical Golgi pattern within 24 h after transfection that colocalized with CGL1-OFP used as an internal control (Fig. 3D, a–c and d–f). This showed that blocking N-glycosylation can restore protein folding of CGL1 C5, independent of the altered amino acid motif, so that migration to Golgi stacks resumes (Supplemental Video S1A). Expression of C5-GFP or C5*-GFP constructs in protoplasts of the stt3a-2 mutant resulted in Golgi labeling within 24 h (Fig. 3D, j–l and m–o), similar to tunicamycin-treated wild-type protoplasts. In the stt3a-2 mutant background, however, signal intensities of Golgi stacks were much lower than in tunicamycin-treated wild-type protoplasts, perhaps because only a small proportion of C5-GFP and C5*-GFP polypeptides are underglycosylated and allowed to exit the ER (Fig. 3D; Supplemental Fig. S3).
In order to biochemically confirm the Golgi localization of CGL1 variants, structures of N-glycans attached to CGL1 polypeptides were analyzed using peptide:N-glycosidase F (PNGase F). PNGase F cleaves N-glycans that do not contain core Fuc modification. Since addition of core Fucs occurs in the Golgi apparatus, PNGase F-resistant N-glycans can be regarded as a hallmark for successful delivery to Golgi stacks. In order to obtain CGL1 variants in sufficient quantity, all constructs were transiently expressed in Nicotiana benthamiana leaves. Subcellular localization and immunoblot profiles of CGL1-GFP variants expressed in agroinfiltrated N. benthamiana leaf tissue were consistent with those expressed in Arabidopsis protoplasts (Supplemental Fig. S4; Supplemental Video S2). We found that C5-GFP displayed a downward mobility shift after PNGase F treatment. The lower Mr form produced after PNGase F digestion migrated similar to nonglycosylated CGL1*-GFP or C5-GFP produced in the presence of tunicamycin. In contrast, CGL1-GFP hardly shifted at all (Fig. 4 ). These findings establish that CGL1-GFP arrives in Golgi stacks, where core fucosylation of its N-glycan occurs, but C5-GFP decorated with two high-Man N-glycans remains in the ER. Taken together, our in planta approaches unequivocally show that CGL1 C5 is likely retained in the ER because of aberrant N-glycosylation. Prevention of aberrant N-glycosylation of C5-GFP and C5*-GFP polypeptides alleviated misfolding in the ER and enabled membrane-bound delivery to Golgi stacks. Next, we set out to determine whether this can explain the restoration of GnTI function in the cgl1 C5 stt3a-2 double mutant.
CGL1 C5-GFP Restores Complex Glycan Maturation in cgl1-T stt3a-2 But Not in cgl1-T The ability of the N-glycosylation variants of CGL1 to restore complex N-glycan production in planta was analyzed by transient expression of CGL1-GFP constructs in cgl1-T mutant protoplasts. In addition, the impact of CGL1 underglycosylation was assessed by expressing the same variants in cgl1-T stt3a-2 protoplasts. In principle, all CGL1-GFP variants expressed in cgl1-T stt3a-2 restored complex glycan formation within 96 h on a fraction of proteins (traveling along the secretory route) compared with mock-transfected controls (minus DNA; Fig. 5 ). The extent of recovery varied among constructs and correlated with expression level of the GFP fusions. Best recovery was observed with CGL1-GFP (wild-type situation) followed by nonglycosylated CGL1*-GFP. These two protein fusions were clearly detectable with GFP antiserum (Fig. 5, arrows in bottom left sections). C5-GFP and C5*-GFP showed delayed and less efficient recovery of complex glycosylation in the cgl1-T stt3a-2 background, consistent with low abundance of the GFP fusions. In cgl1-T mutant protoplasts, only CGL1-GFP and CGL1*-GFP displayed Golgi labeling and produced complex modified glycoproteins (data not shown).
Since all CGL1-GFP variants were localized to the Golgi apparatus in cgl1-T stt3a-2 protoplasts (data not shown), we conclude that complementation depends upon delivery of correctly folded CGL1 variants to Golgi stacks. As already observed for Col wild-type and stt3a-2 mutant protoplasts (Fig. 3B), C5-GFP and C5*-GFP signals were hardly detectable in cgl1-T stt3a-2 protoplasts (Fig. 5, bottom right sections). This might be due to a generally reduced stability of aberrantly glycosylated polypeptides in protoplasts. Nevertheless, this demonstrates that minimal amounts of CGL1 C5 protein are functional and can account for the recovery of complex glycosylation in cgl1 C5 stt3a-2 double mutant lines (Fig. 2B; Supplemental Fig. S2). Probably, this can also explain why transgenic tobacco (Nicotiana tabacum) plants with less than 3% GnTI activity produce normal levels of complex glycans (Strasser et al., 2004 ) and indicates that "low-stainer" plants obtained by an antisense approach in tobacco and potato (Solanum tuberosum; Wenderoth and von Schaewen, 2000) are essentially devoid of GnTI.
None of the tested single or double mutants with N-glycosylation defects displayed obvious growth phenotypes in soil (data not shown). However, differences in root growth were detected on vertical agar plates (Fig. 6
). The most striking morphological deviations (i.e. shorter, more branched, and hairy roots) were displayed by cgl1 C6 stt3a-2 and cgl1-T stt3a-2 double mutants (Fig. 6A). Root growth of cgl1 C5 stt3a-2 seedlings resembled the wild-type and single mutant parents, which can be explained by suppression of the cgl1 C5 defect in the stt3a-2 mutant background (described above). Close-up images of root tips show that all cgl1 alleles produce longer root hairs compared with Col wild type or stt3a-2 (Fig. 6B). In cgl1 C6 stt3a-2 and cgl1-T stt3a-2 double mutants, stunted growth of the root hair zone probably accounts for the bushy appearance (Fig. 6A). Scanning electron microscopy images of root tips show that root hairs of the cgl1-T stt3a-2 double mutant are much thinner and longer than those of the Col wild type (Fig. 6C). The latter seems to be a feature of true loss-of-function cgl1 alleles, since roots of stt3a-2 single and cgl1 C5 stt3a-2 double mutants grown in parallel have shorter hairs (Fig. 6B). Together, these findings point to problems with expansion and probably also division of cells in the root elongation zone as a consequence of combining two basic N-glycosylation defects (i.e. less frequent core glycosylation in the ER combined with aberrant N-glycan modification in the Golgi). An obvious glycoprotein candidate known to affect root tip morphology when mutated in Arabidopsis is KORRIGAN1 (KOR1/RSW2), a class II membrane protein with eight predicted N-glycosylation sites (Nicol et al., 1998
Recovery of complex glycan patterns in the cgl1 C5 stt3a-2 double mutant initially indicated that underglycosylated CGL1 C5 enzyme can rescue the cgl1 defect in Arabidopsis. By studying CGL1-GFP glycosylation variants in Arabidopsis wild type and selected N-glycosylation mutants, we demonstrate that aberrant neoglycosylation also obstructs ER folding of membrane-anchored CGL1 C5 polypeptides in planta. Recovery of complex glycan patterns in the stt3a-2 mutant background showed that the D 144N replacement in CGL1 C5 probably has a less severe effect compared with an equivalent change in rabbit GnTI, which did not create a glycosylation site but substantially affected catalytic activity (Strasser et al., 2005). Analyses of altered N-glycan decoration of CGL1-GFP variants in Arabidopsis protoplasts proved that either no or native glycosylation is compatible with folding of CGL1 polypeptides in the ER and delivery to Golgi stacks. The fact that all CGL1-GFP variants principally complemented the cgl1 defect over time (in protoplasts of the cgl1-T stt3a-2 double mutant) supports the notion that low amounts of functional GnTI suffice to guarantee complex glycosylation in plants. Since no deviant phenotype was detected in aerial parts of true cgl1 stt3a double mutants, we reasoned that combination of two basic N-glycosylation defects might influence only a subset of all glycoproteins synthesized during certain stages of Arabidopsis development. Indeed, stunted root growth and altered root hair morphology indicate that among the glycoproteins synthesized during root cell elongation are prime targets affected.
STT3 is a proposed catalytic subunit of OST and encoded by paralogous genes in Arabidopsis. Although our recent study indicates that STT3a plays a predominant role in N-glycosylation of KOR1 (Kang et al., 2008
According to the analyses presented here, cgl1 C5 defines a conditional Arabidopsis allele and only cgl1 C6 and cgl1-T can be considered true loss-of-function mutants. It is conceivable that the leakiness of the cgl1 C5 mutation becomes relevant under stressful conditions (Kang et al., 2008 Finally, our data demonstrate that small amounts of functional GnTI protein are sufficient to produce nearly wild-type levels of complex glycans in plants. Considering that conventional gene silencing often does not yield complete loss of expression, more robust strategies (e.g. double-stranded RNA interference) are needed to study the role of reduced complex glycosylation in crop plants.
Plant Growth
Arabidopsis (Arabidopsis thaliana Col) plants were grown in soil under a long-day regime (16 h of light, 21°C). Backcrossed lines (more than seven rounds) of cgl1 C5#5 and cgl1 C6#22 EMS mutants (von Schaewen et al., 1993 For protoplast isolation, Arabidopsis seeds were surface sterilized with 12% hypochloride, placed on agar medium (0.8% agar, 1% Suc, and 0.5 Murashige and Skoog [MS] salts plus vitamins, pH 5.8), and kept at 4°C for 3 d. Plates were transferred to a growth room with artificial illumination (Phillips TLD, 36W/827 and 36W/840 twin bulb sets). One-week-old seedlings were transferred to Magenta boxes (Sigma) and grown in sterile culture for 4 to 5 weeks under a long-day regime (16 h of light/21°C, 8 h of dark/18°C) prior to harvesting sterile leaf material.
For root growth analyses, Arabidopsis seeds were surface sterilized, placed on hard agar plates (1.5% agar, 3% Suc, and MS salts plus vitamins, pH 5.8), and stratified for 3 d (Koiwa et al., 2003
Nicotiana benthamiana plants were grown in soil in the greenhouse with permanent fertilization (2
Genomic DNA was isolated from small amounts of leaf tissue (Dilworth and Frey, 2000
Total RNA from leaf tissue was isolated with the guanidinium HCl procedure (Logemann et al., 1987
To obtain a three-dimensional structure model, the amino acid sequence of Arabidopsis CGL1 was analyzed with Swiss-model, an automated comparative protein-modeling server at http://swissmodel.expasy.org/workspace. A glycosylated protein model of CGL1 was created with GlyProt for in silico glycosylation of proteins (Glycosciences.de of the Deutsches Krebsforschungszentrum Heidelberg) at http://www.glycosciences.de/modeling/glyprot/php/main.php.
For OFP fusion constructs, we started from pOFP-
Protoplasts were prepared from leaves of 4- to 5-week-old Arabidopsis plants in sterile culture and transfected with the polyethylene glycol method as described by Damm et al. (1989)
Agro-leaf infiltration of soil-grown N. benthamiana plants was essentially done as described by Schöb et al. (1997)
Light microscopy was performed with a confocal laser-scanning microscope (Leica TCS SP2/AOBS) set up for simultaneous three-channel color detection. Fluorescing cells were spotted under an inverse microscope (Leica DM IRE2 UV) equipped with filter sets for detecting GFP (L5; Leica) or OFP (Phycoerythrin; AF Analysentechnik). One of two water-dipping objectives was used: 63x (0.9 W) for submerged leaf tissue on Petriperm plates (flexible bottom; Greiner), or 63x (1.2 W corrected) for protoplasts in a Bachhofer chamber (coverslip bottom). Confocal laser-scanning microscopy images were recorded digitally using the Leica confocal software. Excitation/emission wavelengths were 488/490 to 510 nm for GFP and 543/580 to 600 nm for OFP (orange-shifted mRFP or DsRed). As a reference, chloroplast fluorescence was recorded above 700 nm. During coexpression analyses, care was taken that real-time recorded signals did not bleed through into other detection channels.
Equal amounts of cultivated protoplasts were cooled on ice and harvested by centrifugation (5 min at 1,000g, 4°C). Supernatants were discarded and cell pellets were frozen in liquid nitrogen. Leaf discs were excised with a cork borer and immediately frozen in liquid nitrogen. Concomitant with thawing in protein extraction buffer (50 mM HEPES-NaOH, pH 7.5, 250 mM NaCl, 1 mM EDTA, 1% β-mercaptoethanol, 0.6% SDS, and 1 mM Pefabloc SC; Serva), a tip of spatula polyvinylpolypyrrolidone (Sigma) was added to prevent protein oxidation. Samples were ground with a potter, and cleared supernatants were used for further analyses. Prior to PNGase F treatment, cleared leaf extracts were boiled for 3 to 5 min. Heated samples were diluted with extraction buffer to reduce SDS to 0.2% (f.c.) and supplemented with Triton X-100 (1% f.c.) and EDTA (10 mM f.c.). One unit of PNGase F (Roche) was added to each sample, mixed, and kept at 37°C for 18 h or longer prior to SDS-PAGE. Mock-incubated samples were treated in the same way, except for omitting the enzyme. Successful PNGase F digests reduce concanavalin A binding to blotted glycoproteins in the presence of Ca2+ and Mn2+ ions, as revealed by peroxidase-coupled affinoblot detection (Faye and Chrispeels, 1985
Protein extracts were separated by SDS-PAGE followed by western-blot transfer using the MiniProtean-III system (Bio-Rad). In brief, SDS gels were rinsed with transfer buffer (25 mM Tris, 192 mM Gly, and 5% methanol) and electrotransferred to a nitrocellulose membrane (PROTRAN; Schleicher & Schüll). Blotted proteins were stained with Ponceau S (0.3% [w/v] in 3% TCA; Serva) on the blot, documented with a scanner, and destained with TBST (20 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) prior to 1-h incubation of the membrane in blocking buffer (2% nonfat milk powder in TBST). Incubation in primary antibodies diluted in blocking buffer (1:300–1:50,000 depending on the antiserum and detection system used) was either at room temperature for 2 h or at 4°C overnight. Blots were washed with TBS (20 mM Tris-HCl, pH 7.4, 500 mM NaCl), TBST, and TBS (10 min each) and incubated for 1 h with goat anti-rabbit horseradish peroxidase conjugate (Bio-Rad), diluted 1:3,000 in blocking buffer for colorimetric detection, where indicated (von Schaewen et al., 1993
Root growth assays were conducted on hard agar plates as described previously (Koiwa et al., 2003
Seedlings were removed from hard agar plates and taken through a series of dehydration steps with increasing ethanol concentrations. Seedlings were kept in absolute ethanol, then dried with a critical point drier (Emitech K850), gold coated with a vacuum-sputter device (Emitech K550x), and examined by scanning electron microscopy (Hitachi S-3000N) at 15 kV under high vacuum. Micrographs were recorded digitally.
The following materials are available in the online version of this article.
We are grateful to Klaus Tenberge (Institute for Botany, Westfälische Wilhelms-Universität Münster) for basic instructions on scanning electron microscopy. Several colleagues shared their research material with us. We thank Benedikt Kost (University of Warwick, United Kingdom) for plasmid vector pGFP2, Shaoul Yalovsky (Tel Aviv University, Israel) for plasmid vector pSY526, and Sarah Hodge (Medical Research Council, Cambridge, United Kingdom) for pBIN m-gfp5-ER. KOR1-specific antibodies were provided by the group of Herman Höfte (INRA, Versailles, France), and Agrobacterium strain 19K was provided by the group of Jörg Kudla (Institute for Botany, Westfälische Wilhelms-Universität Münster). This study received excellent technical support from Kerstin Fischer, Olessja Becker, Meenu Vikram, and Jae Sook Kang. Received July 25, 2008; accepted August 24, 2008; published September 3, 2008.
1 This work was supported by the Deutsch Forschungsgemeinschaft (grant nos. SCHA 541/7 and HBFG 112–480 to A.v.S.), by the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service Designing Food for Health Program (grant no. 2008–34402–19195 to H.K.), and by the Texas AgriLife Research Federal Initiative (grant no. 2007–118409 to H.K.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Antje von Schaewen (schaewen{at}uni-muenster.de).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.127027 * Corresponding author; e-mail schaewen{at}uni-muenster.de.
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ABSTRACT
RESULTS AND DISCUSSION
1,3-Fuc and β1,2-Xyl residues. GnTI shows class II membrane-protein topology, consisting of the N-terminal cytosolic domain followed by a single-span transmembrane domain (or membrane anchor), hinge region, and a large C-terminal luminal domain for enzymatic activity. In accordance with their proposed location, GnTI-GFP fusion proteins were shown to label Golgi stacks in plant cells (Essl et al., 1999
NPK 14:6:24) and additional illumination (12 h of light/22°C; Phillips 400W/645 Master HPI-T Plus light bulbs, 250 µE at plant height). Three-week-old plants were used for Agro-leaf infiltrations. 
Nco (T. Meyer, personal communication), a derivative of plant expression vector pSY526 with cauliflower mosaic virus 35S-mRFP cassette. The second NcoI site in the mRFP coding region was eliminated by silent mutagenesis using the Quick Change PCR mutagenesis protocol (Stratagene) and Phusion High-Fidelity DNA Polymerase (Finnzymes). The OFP-ER control was obtained by multistep cloning. First, a 950-bp HindIII-EcoRI fragment, comprising the 35S promoter and signal sequence of pBIN m-gfp5-ER (Haseloff et al., 1997