Accepted Article Received Date : 07-Jul-2014 Accepted Date : 11-Sep-2014 Article type : Original Article The transmembrane domain of N-acetylglucosaminyltransferase I is the key determinant for its Golgi sub-compartmentation Jennifer Schoberer1, Eva Liebminger1, Ulrike Vavra1, Christiane Veit1, Alexandra Castilho1, Martina Dicker1, Daniel Maresch2, Friedrich Altmann2, Chris Hawes3, Stanley W. Botchway4 and Richard Strasser1, * 1 Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria 2 Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria 3 Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, United Kingdom 4 Research Complex at Harwell, Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot OX11 0QX, United Kingdom This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12671 This article is protected by copyright. All rights reserved. Accepted Article *Corresponding author: R. Strasser, Tel: 43-1-47654-6705; Fax: 43-1-47654-6392; E-mail: richard.strasser@boku.ac.at Running Title: GnTI CTS domain function Total Keywords:, Golgi apparatus, Golgi targeting, Golgi retention, N-glycan processing, glycosyltransferase, type II membrane protein, protein-protein interaction, transmembrane domain, Arabidopsis thaliana, Nicotiana benthamiana Summary Golgi-resident type II membrane proteins are asymmetrically distributed across the Golgi stack. The protein intrinsic features that determine the sub-compartment specific protein concentration are still largely unknown. Here, we used a series of chimeric proteins to investigate the contribution of the cytoplasmic, transmembrane and stem region of tobacco N-acetylglucosaminyltransferase I (GnTI) for its cis/medial-Golgi localization and for protein-protein interaction in the Golgi. The individual GnTI protein domains were replaced with the ones from the well-known trans-Golgi enzyme α2,6sialyltransferase (ST) and transiently expressed in Nicotiana benthamiana. Using co-localization analysis and N-glycan profiling, we show that the transmembrane domain of GnTI is the major determinant for its cis/medial-Golgi localisation. By contrast, the stem region of GnTI contributes predominately to homomeric and heteromeric protein complex formation. Importantly, in transgenic Arabidopsis thaliana, a chimeric GnTI variant with altered sub-Golgi localisation was not able to complement the GnTI-dependent glycosylation defect. Our results suggest that sequence-specific features in the transmembrane domain of GnTI account for its steady-state distribution in the cis/medial-Golgi in plants, which is a prerequisite for efficient N-glycan processing in vivo. This article is protected by copyright. All rights reserved. Accepted Article Introduction The Golgi apparatus is the central biosynthetic organelle of the secretory pathway. It receives cargo proteins, polysaccharides and lipids from the endoplasmic reticulum (ER), subjects them to extensive processing in different sub-compartments and transports the cargo to other destinations within the endomembrane system. The compartmentation of biosynthetic activities in different cisternae of a polarised Golgi stack is a major function of the Golgi. Many processing steps involve modifications of protein- or lipid-bound oligosaccharides which are carried out by a large number of Golgi-resident glycosyltransferases and glycosidases. The overlapping but non-uniform distribution of these glycosylation enzymes across the Golgi stack is well documented and has been shown for plants by immunoelectron and confocal microscopy (Chevalier et al., 2010; Reichardt et al., 2007; Saint-JoreDupas et al., 2006; Schoberer et al., 2010). The specialized Golgi architecture provides an excellent means to asymmetrically distribute these enzymes and consequently ensure the sequential order of glycan processing on transiting cargo. The concentration of Golgi glycosylation enzymes in distinct Golgi-domains is a prerequisite for controlled glycan biosynthesis as different glycosyltransferases and glycosidases may compete for identical substrates and certain reaction products inhibit the action of other enzymes resulting in partially processed glycans. The removal of mannose residues from hybrid N-glycans by Golgi-α-mannosidase II, for example, is blocked by prior action of β1,4galactosyltransferase (Bakker et al., 2001; Palacpac et al., 1999). Yet, despite our understanding of the functional importance of compartmentation of Golgi glycosylation enzymes the signals and underlying mechanisms required to establish and maintain the asymmetric distribution are still largely unknown in plants and other organisms (Oikawa et al., 2013; Schoberer and Strasser, 2011; Tu and Banfield, 2010). The dynamic distribution and trafficking of resident proteins in the Golgi is dependent on the overall cargo transport mechanism through this organelle, which is still controversial. The constant flux of cargo and the dynamic distribution of Golgi-integral membrane proteins suggest that the polar distribution of Golgi-resident proteins is achieved by a coordinated interplay of retrieval and This article is protected by copyright. All rights reserved. Accepted Article retention mechanisms. The majority of the Golgi glycosyltransferases and glycosidases are type II membrane proteins consisting of a short cytoplasmic tail, a single transmembrane region, a flexible stem and a large catalytic domain that faces the lumen of the Golgi cisternae (Schoberer and Strasser, 2011). This basic domain organisation is similar between Golgi enzymes from different eukaryotic kingdoms and the underlying Golgi-targeting/retention or retrieval mechanisms seem also highly conserved between species (Bakker et al., 2001; Boevink et al., 1998; Wee et al., 1999; Palacpac et al., 1999). Consequently, these enzymes should either contain a specific amino acid sequence motif or protein conformation that leads to the observed steady-state localization within the Golgi apparatus. For Golgi-localization and sorting of glycosylation enzymes different mechanisms have been proposed. The oligomerization or kin recognition model is based on the possibility that glycosylation enzymes can form homo- or heteromeric protein complexes that are together retained or concentrated in distinct regions of the Golgi apparatus (Machamer, 1991; Nilsson et al., 1994). For N-glycan processing enzymes distinct Golgi protein complex formation has been described in mammalian cells as well as in plants (Hassinen et al., 2010; Schoberer et al., 2013). By contrast, the bilayer thickness model postulates that changes in the thickness of the lipid bilayer restrict the forward transport of proteins with shorter transmembrane domains and thus could play an important role in retention of proteins in different Golgi cisternae (Bretscher and Munro, 1993). Experimental evidence revealed that the transmembrane domain length and/or sequence composition are important determinants of subcellular distribution in different eukaryotes (Brandizzi et al., 2002a; Munro, 1995; Saint-Jore-Dupas et al., 2006). These studies examined mainly the contribution of the transmembrane domain length in Golgi retention in comparison to other organelles such as the ER and plasma membrane, but the impact on sub-Golgi localization has not been addressed in detail. In addition, recent studies from yeast have highlighted a role of the short cytoplasmic tail of glycosylation enzymes as a determinant of Golgi-retention and intra Golgi-trafficking (Schmitz et al., 2008; Tu et al., 2008). In this receptor-mediated retrieval model the coat protein I complex (COPI) This article is protected by copyright. All rights reserved. Accepted Article binds via the peripheral membrane protein Vps74p to a specific amino acid sequence stretch in the cytoplasmic tail of glycosyltransferases, leading to Golgi retention or retrograde trafficking. In plants, the interaction of the cytoplasmic tail of the multi-pass transmembrane protein EMP12 with COPI maintains its Golgi retention (Gao et al., 2012), but the impact of the cytoplasmic tail on Golgilocalization of type II membrane proteins is unclear. Here, we addressed the question whether such mechanisms involving either the cytoplasmic tail or the transmembrane domain or the stem region are responsible for the steady-state Golgi distribution of tobacco N-acetylglucosaminyltransferase I (GnTI). GnTI is a cis/medial-Golgi-resident type II membrane protein that plays a key role in N-glycan processing, because it initiates the formation of complex N-glycans in animals and plants (Burke et al., 1994; Schoberer et al., 2009). We focused on the N-terminal cytoplasmic-transmembrane and stem (CTS)-region of GnTI which is sufficient for subGolgi localization in leaves of N. benthamiana plants and performed domain-swap experiments. The cytoplasmic tail, transmembrane or stem region of GnTI were exchanged with the corresponding regions from rat α2,6-sialyltransferase (ST) which is the most widely used trans-Golgi marker in plants (Boevink et al., 1998). We examined the contribution of the individual protein regions to their subcellular localization, protein complex formation and ability to restore N-glycan processing in the A. thaliana gntI mutant. Our data provide insights for the specific role of individual GnTI domains in Golgi localization and subsequent in vivo function in plants. Results N-glycan analysis demonstrates differences in subcellular localization of chimeric type II membrane proteins To examine the role of the N-terminal region in sub-Golgi localization of tobacco GnTI we generated reporter constructs consisting of chimeric CTS regions from GnTI (NNN) and ST (RRR). We chose the Golgi targeting domains (Figure 1a) from these two glycosyltransferases because they lead to an This article is protected by copyright. All rights reserved. Accepted Article overlapping, but distinct sub-Golgi distribution when transiently expressed in leaves of N. benthamiana and their CTS regions do not physically interact (Schoberer et al., 2013; Schoberer et al., 2010). Six chimeric proteins were designed by exchanging the respective cytoplasmic tail, transmembrane domain and luminal stem region (Figure 1b). In our first approach, we fused all six chimeric CTS regions to a glycosylation reporter (GFPglyc) consisting of the IgG1 heavy chain fragment (Fc-domain) and GFP (Figure 1c) (Schoberer et al., 2009). The Fc-domain is used for affinity purification of expressed proteins and contains a single N-glycosylation site that can be utilized to monitor differences in N-glycan processing. To analyze whether the different chimeric CTS regions lead to differences in subcellular localization the CTS-GFPglyc variants were transiently expressed in leaves of N. benthamiana. Purified CTS-GFPglyc proteins were trypsin-digested and peptides were subjected to MS analysis. The N-glycosylation profile of NNN-GFPglyc and RRR-GFPglyc displayed almost exclusively a peak corresponding to the complex N-glycan GlcNAc2XylFucMan3GlcNAc2 (GnGnXF) which confirms processing in the Golgi apparatus. Peaks representing incompletely processed or further elongated N-glycan structures were only found in low amounts (Figure 2). The GnGnXF Nglycan was also detected as predominant structure on NNR-GFPglyc, RNR-GFPglyc, RNN-GFPglyc and NRR-GFPglyc. By contrast, the N-glycan analysis of NRN-GFPglyc and RRN-GFPglyc revealed primarily peaks corresponding to oligomannosidic N-glycans (Man5GlcNAc2 to Man9GlcNAc2) (Figure 2) and a peak corresponding to the unglycosylated peptide (Figure S1). The oligomannosidic structures are indicative of retention in the ER and a similar profile was also detected on the ER-retained reporter GCSI-GFPglyc. In vivo protein galactosylation reveals differences in Golgi sub-compartmentation of chimeric CTS region containing proteins Data from previous studies suggest that the attachment of β1,4-linked galactose to N-glycans in the Golgi can be used to monitor differences in sub-Golgi localization (Bakker et al., 2001; Bakker et al., This article is protected by copyright. All rights reserved. Accepted Article 2006; Palacpac et al., 1999; Strasser et al., 2009). N-glycans with β1,4-linked galactose residues are normally not present in plants and the responsible β1,4-galactosyltransferase (GALT) competes with other N-glycan processing enzymes for the acceptor substrates. As a consequence of β1,4galactosylation, the access of endogenous Golgi-resident enzymes like Golgi-α-mannosidase II (GMII) to their substrates is blocked resulting in the formation of incompletely processed N-glycans (Figure S2). We hypothesized that co-expression of chimeric CTS-GALT enzymes leads to alterations in Nglycosylation dependent on the sub-Golgi distribution of CTS-GALT. To test our approach we fused the CTS regions from GnTI and ST to the catalytic domain of Homo sapiens GALT and analyzed the generated N-glycans of a co-expressed monoclonal antibody (mAb) which served as a glycoprotein reporter (Strasser et al., 2009). The mAb N-glycan profile obtained by fusion of GALT to the CTS region of the cis/medial-Golgi enzyme GnTI were unambiguously different from the one derived by RRR-GALT (Figure 3a). The glycopeptide profile obtained by co-expression of NNN-GALT consisted mainly of incompletely processed structures (Man5, Man4A/Man5Gn, Man5A). By contrast, these structures were less abundant when the trans-Golgi targeting region RRR was fused to GALT. In that case, complex galactosylated N-glycan structures containing xylose and fucose residues (e.g. MAXF, GnAXF, AAXF) were more abundant (Figure 3a). Next, we fused the chimeric CTS regions to the catalytic domain of GALT and co-expressed them with the glycoprotein reporter. The N-glycans co-expressed with RNR-, RNN- and NNR-GALT displayed primarily incompletely processed N-glycans being indicative of cis/medial-Golgi localization (Figure 3b). NRR-GALT generated more fully galactosylated complex N-glycans and thus resembles transGolgi targeting. Consistent with the previously detected ER-retention (Figure 2), NRN-GALT and RRNGALT did not produce significant amounts of galactosylated N-glycans and the N-glycan profile was comparable to the one from mAb without any co-expressed GALT or from the ER-retained version GCSI-GALT (Figure 3a, b). Collectively, these data strongly indicate that the chimeric RNR, RNN and NNR CTS regions concentrate proteins mainly in the cis/medial-Golgi while NRR mediates predominately trans-Golgi accumulation. This article is protected by copyright. All rights reserved. Accepted Article The transmembrane domain of GnTI plays an important role for its sub-Golgi localization To further investigate the contribution of the individual domains to sub-Golgi localization, we analyzed the subcellular localization of chimeric CTS-GFPglyc variants by live-cell confocal microscopy. As expected, NNR-, RNR-, RNN- and NRR-GFPglyc marked the Golgi (Figure 4). In agreement with our data from N-glycan analysis, NRN-GFPglyc displayed mainly ER-labelling and RRN-GFPglyc showed ER localization as well as targeting to other subcellular compartments like the cytoplasm (Figure 4). Next, we used confocal microscopy to determine the sub-Golgi distribution of the chimeric CTSGFPglyc proteins in comparison with the cis/medial-Golgi located Golgi matrix protein AtCASP-mRFP (Osterrieder et al., 2009; Renna et al., 2005; Schoberer et al., 2010). The fluorescence profiles for chimeric CTS-GFPglyc and AtCASP-mRFP across Golgi stacks revealed clear differences for NRR, while NNR, RNN and RNR shifted to a lesser extent (Figure 5a). To more precisely analyze the sub-Golgi localization we calculated the Pearson’s correlation coefficient for co-localization with AtCASP-mRFP and the trans-Golgi marker RRR-mRFP. While the correlation between NRR-GFPglyc and AtCASP-mRFP was substantially lower than for NNN-GFPglyc and AtCASP-mRFP, the NNR, RNR and RNN correlation was more similar to NNN (Figure 5b and Figure S3). By contrast, NRR-GFPglyc displayed a strong correlation with RRR-mRFP. On the other hand, NNR, RNR and RNN displayed like NNN a significantly lower correlation. Consistent with the N-glycan analysis, these data highlight that the transmembrane domain plays an important role for cis/medial-Golgi localization of GnTI, while the cytoplasmic tail and stem region are not involved in sub-Golgi distribution. The stem region of GnTI is relevant for homo- and heterodimer formation In a previous study, we have demonstrated that tobacco GnTI forms homodimers in the Golgi apparatus, which is mediated by the N-terminal CTS region (Schoberer et al., 2013). To test the contribution of the different domains to protein-protein interaction, we co-expressed NNN-GFPglyc with mRFP-tagged chimeric CTS regions (RNR, NRR, RNN and NNR) in N. benthamiana leaves and This article is protected by copyright. All rights reserved. Accepted Article purified GnTI-GFPglyc by binding to Protein A. Immunoblot analysis revealed that the amount of copurified RNN-mRFP was similar to NNN-mRFP, while binding of NNR-mRFP, RNR-mRFP and NRRmRFP was as low as RRR-mRFP (Figure 6a) which does not interact with GnTI-GFPglyc (Schoberer et al., 2013). Similarly, when NNN-mRFP was co-expressed with chimeric CTS-GFPglyc interaction was only found for RNN (Figure 6b). In addition, when MNS1-GFPglyc, which forms a heteromeric complex with GnTI (Schoberer et al., 2013) was used to co-purify chimeric CTS-mRFP proteins, considerable amounts of the heteromeric MNS1/RNN complex were detected (Figure 6c). To examine whether the catalytic domain plays any role in complex formation we fused the chimeric RNR region to the full-length catalytic domain of tobacco GnTI (RNR-GNTI-GFP), co-expressed RNRGNTI-GFP with the control NNN-GNTI-mRFP (GnTI CTS region fused to the catalytic domain) and performed co-immunoprecipitation followed by immunoblot detection. In agreement with our previous data, no marked interaction could be found between RNR-GNTI-GFP and NNN-GNTI-mRFP (Figure 6d). Collectively, the co-IP experiments suggest that the GnTI stem region is primarily required for complex formation. To verify the co-IP results and test for direct interaction of the individual domains, we selected specific chimeric CTS-mRFP fusions and tested the in vivo GnTI interactions using two-photon excitation FRET-FLIM (Schoberer et al., 2013). The average excited-state fluorescence lifetime of the NNN-GFPglyc donor was 2.44 ns in the absence of an acceptor fluorophore (Table 1). The presence of co-expressed NNN-mRFP led to a significant quenching of the donor lifetime to an average of 2.08 ns (14.56% FRET efficiency), which is indicative of a strong protein-protein interaction. Similarly, coexpression with RNN-mRFP produced FRET efficiency values of 10.79%, which indicates physical interaction and dimer formation in the Golgi membrane. By contrast, donor quenching was less efficient in the presence of RNR-mRFP indicating no or only a weak interaction and the values obtained in the presence of NRR-mRFP were in the range of those for RRR-mRFP, which does not This article is protected by copyright. All rights reserved. Accepted Article physically interact with GnTI (Schoberer et al., 2013). Taken together, the FRET-FLIM data are consistent with the co-IP results and highlight the importance of the stem region in GnTI homodimer formation. Complementation of the N-glycan processing defect requires correct sub-Golgi targeting signals Next, to examine whether the findings obtained from the chimeric reporter proteins can be applied to full-length GnTI and its in vivo N-glycan processing activity we generated transgenic A. thaliana gntI plants expressing the chimeric CTS regions fused to the catalytic domain of A. thaliana GnTI (AtGNTI). To exclude any overexpression effect the chimeric AtGNTI proteins were expressed under the control of the endogenous GnTI promoter. The complementation of the N-glycan processing defect of gntI plants was analyzed by immunoblotting of protein extracts with antibodies directed against complex N-glycans. As expected, AtNNN-AtGNTI complemented the N-glycan processing defect of gntI and restored complex N-glycan formation (Figure 7a). By contrast, RRR-AtGNTI expression did not rescue the N-glycan processing defect suggesting that the ST-mediated trans-Golgi targeting of GnTI is not functional. Consistent with an altered steady-state sub-Golgi distribution, NRR-AtGNTI expressing gntI plants did not produce complex N-glycans (Figure 7b). On the other hand, RNN-AtGNTI, RNR-AtGNTI and NNR-AtGNTI were functional and rescued the complex N-glycan processing defect. In summary, our data indicate that distinct domains within the CTS region are crucial for the sub-Golgi localization and subsequently for the in vivo function of GnTI in plants. Discussion A central biosynthetic function of the Golgi is the modification of protein and lipid-bound glycans and polysaccharides. Typically, this function is carried out by type II membrane proteins that are asymmetrically distributed in some kind of assembly line across the Golgi stack. In yeast and This article is protected by copyright. All rights reserved. Accepted Article mammalian cells, different protein regions have been found to contribute to Golgi localization of glycan modifying enzymes (Fenteany and Colley, 2005; Grabenhorst and Conradt, 1999; Schmitz et al., 2008; Tu et al., 2008). In contrast, the sub-Golgi targeting determinants of most glycosyltransferases and glycosidases are largely unknown. Dependent on the mode of cargo transport through the Golgi these domains contain either retention signals (vesicular transport model) or retrograde trafficking signals (cisternal maturation model) (Rabouille and Klumperman, 2005). Here, we tested the contribution of the different domains from the N-terminal Golgi-targeting region of the cis/medial-Golgi enzyme GnTI for sub-Golgi localization. To obtain detailed information on sub-Golgi targeting we used live-cell imaging (Figures 4 and 5) and took advantage of sensitive biochemical approaches based on the monitoring of changes in N-glycan processing (Figures 2 and 3). Analysis of the N-glycan profile of a chimeric glycoprotein (Figure 2) provided information on the topology of the expressed proteins as only correctly orientated forms are glycosylated in the lumen of the ER and allowed us to discriminate between ER-retention and Golgi-targeting. Interestingly, the N-glycan modifications appeared independent of the cis/medial- or trans-Golgi targeting regions indicating that the dynamic distribution of N-glycan processing enzymes leads to contact of cargo with processing enzymes from other Golgi cisternae. However, due to substrate competition the impact on sub-Golgi compartmentation was clearly discernible when chimeric CTS regions were fused to the catalytic domain of GALT (Figure 3). Strikingly, most Golgi-resident type II membrane proteins contain a short tail that faces the cytoplasm. In mammals as well as in plants, the short cytoplasmic region contains a basic amino acid motif that is required for COPII vesicle interaction and ER export (Giraudo and Maccioni, 2003; Schoberer et al., 2009). Earlier studies with mammalian glycosyltransferases showed that these cytoplasmic tails are also implicated in sub-Golgi localization which could be mediated by binding of cytosolic proteins (Uliana et al., 2006). For rat ST as well as for human GnTI it has been proposed that the cytoplasmic domain contributes to Golgi-localization (Burke et al. 1994; Fenteany and Colley, This article is protected by copyright. All rights reserved. Accepted Article 2005). However, our data from swapping of the cytoplasmic tails clearly show that these N-terminal amino acid regions are not involved in cis/medial- or trans-Golgi concentration of GnTI and ST in plants (Figures 3 and 5). Moreover, the GnTI variant with the cytoplasmic tail from ST was fully functional in vivo (Figure 7). In yeast, the peripheral Golgi protein Vps74p interacts with motifs in the cytoplasmic tails of glycosyltransferases and subsequently functions as a glycosyltransferase sorting receptor for their retrograde trafficking and/or Golgi retention (Schmitz et al., 2008; Tu et al., 2008). Further studies revealed that GOLPH3, the mammalian Vps74p ortholog, interacts with a conserved amino acid sequence motif present in the cytoplasmic tail of distinct glycosyltransferases (Ali et al., 2012). Interestingly, A. thaliana and other plants seem to lack Vps74p/GOLPH3 homologs and so far, a conserved sequence motif could not be detected in the cytoplasmic tail of plant type II membrane proteins (Schoberer and Strasser, 2011) which suggests that there are fundamental differences in the mechanisms that concentrate glycan modifying type II membrane proteins in plants and in other kingdoms. Using time-resolved fluorescence imaging we recently detected the formation of homo- and heterodimers between N-glycan processing enzymes located in the early Golgi (Schoberer et al., 2013). The organization in multi-protein complexes might contribute to their Golgi localization and/or modulate their activity. It was previously proposed that the oligomerization or kin recognition of glycosylation enzymes in mammals is important for Golgi retention by excluding large multienzyme complexes from vesicles that mediate cargo transport (Machamer, 1991). For human GnTI the formation of homodimers has been described and it has been suggested that oligomerization plays a major role for Golgi retention (Hassinen et al., 2010). In line with data for mammalian GnTI (Nilsson et al., 1996), we observed that the stem region of tobacco GnTI is involved in homomeric and heteromeric complex formation (Figure 6). However, our data indicate also that the proteinprotein interaction is not implicated in sub-Golgi compartmentation. In the absence of a strong interaction the sub-Golgi localization of Golgi-resident GnTI-chimeras appears not considerably This article is protected by copyright. All rights reserved. Accepted Article altered suggesting that the complex formation is not a prerequisite for cis/medial-Golgi concentration of GnTI. Interestingly, the almost full restoration of complex N-glycan formation in transgenic gntI plants expressing chimeras that display no or weak protein-protein interaction (Figure 7) hints that a kin recognition process plays only a minor role for the functionality of GnTI. Nonetheless, it cannot be excluded that homomeric or heteromeric protein complexes are required to modulate or fine-tune the activity of GnTI. Such subtle modifications might be required in certain cell-types or under adverse environmental conditions (Kang et al., 2008). Enhanced in vitro enzyme activity due to complex formation has for example been demonstrated for for plant glycosyltransferases involved in arabinogalactan biosynthesis (Dilokpimol et al., 2014). Moreover, there is emerging experimental evidence that complex formation of Golgi-resident proteins occurs quite frequently in plants (Oikawa et al., 2013). However, the biological relevance of these complexes in modulation of enzyme activities or substrate specificities and finally in regulation of glycan biosynthesis remains to be shown. The transmembrane domain is implicated in sub-Golgi localization of tobacco GnTI An important role of the transmembrane domain for Golgi retention was described for mammalian N-glycan processing enzymes such as GALT and ST (Munro, 1995; Nilsson et al., 1991). For a chimeric protein containing the transmembrane domain of rabbit GnTI, only partial Golgi retention was described indicating that several regions cooperatively mediate its Golgi localization (Burke et al., 1994). Our data provide evidence that the transmembrane domain is the key determinant for subGolgi distribution of plant GnTI. The chimeras containing the GnTI transmembrane domain flanked by ST regions were predominately found in the same compartment as GnTI (Figures 3 and 5) and importantly, the RNR-AtGNTI chimeric protein was functional when expressed under native conditions in A. thaliana (Figure 7). By contrast, however, the role of the transmembrane domain for targeting is less clear for ST (Table 2). While RRR and NRR are found in the trans-Golgi, NRN and RRN This article is protected by copyright. All rights reserved. Accepted Article are seen in the ER and in the cytoplasm. The Golgi targeting of ST in plants might therefore either require additional protein domains like the stem region or the chimeric NRN and RRN proteins display aberrant features that are recognized by the ER quality control system. In the bilayer thickness model it was proposed that the length of the hydrophobic domain of glycosyltransferases could be implicated in sorting (Bretscher and Munro, 1993). This model is based on the finding that ER/Golgi-resident proteins tend to have a shorter transmembrane domain than plasma membrane proteins and the observation that the bilayer length and composition is not homogenous throughout the endomembrane system. Hence, proteins with shorter hydrophobic stretches could be excluded from incorporation into thicker membrane regions leading to a partitioning into different domains. For example, an increased plasma membrane expression of type I protein chimeras was found when additional residues were inserted into the transmembrane domain. (Brandizzi et al., 2002a). A seven amino acid increase of the transmembrane region of soybean Golgi-α-mannosidase I caused a shift from the cis/medial- to the trans-Golgi (Saint-JoreDupas et al., 2006) indicating that the length of the hydrophobic stretch or the presentation of certain amino acids from the transmembrane domain could be essential factors for its sub-Golgi targeting. Our finding that the transmembrane domain is the major determinant for Golgi subcompartmentation of GnTI is consistent with such lipid-based sorting processes. However, the predicted length of the transmembrane spanning regions from tobacco GnTI and ST are almost identical, which makes it unlikely that the number of amino acids alone contributes to the specific sub-Golgi concentration. Apart from the length of the transmembrane domain, we propose therefore that the amino acid composition could play a major role in sub-Golgi localization of type II membrane proteins. We and others have previously compared the length and the composition of the transmembrane domains of different Golgi resident plant N-glycan processing enzymes and did not find any consensus sequence motif that distinguishes cis/medial- from trans-Golgi enzymes (Nikolovski et al., 2012; Saint-Jore-Dupas et al., 2006; Schoberer and Strasser, 2011; van Dijk et al., 2008). The low number of plant type II membrane proteins with confirmed sub-Golgi localization This article is protected by copyright. All rights reserved. Accepted Article precludes a thorough comparison of sequence features. However, a more comprehensive bioinformatic approach could also not delineate a conserved sequence motif responsible for subGolgi targeting in a large number of mammalian proteins (Sharpe et al., 2010). While organellespecific properties might discriminate between ER, Golgi and plasma membrane localization (Nikolovski et al., 2012; Sharpe et al., 2010) it is likely that Golgi sub-compartmentation of individual proteins is determined by different protein intrinsic characteristics rather than by a single mechanism. In summary, we show in this study that the sub-Golgi localization is crucial for in vivo functionality of GnTI. While it appears that the cis-/medial Golgi concentration of GnTI is mediated by the transmembrane domain, the homo- and heterodimer formation is strongly dependent on the stem region. Our data show that this protein-protein interaction is less important for sub-Golgi compartmentation of GnTI and its in vivo function. However, further studies are required to analyze the biological significance of the complexes and the interaction with other Golgi-resident proteins from the same or different biosynthetic pathways. Eventually, these studies will lead to a better understanding of mechanisms that govern protein homeostasis and function of glycan-modifying enzymes in the Golgi. Experimental procedures Cloning of constructs The RNN expression construct was generated by ligation of two overlapping synthetic oligonucleotides (RSTC_1F/2R) (Table S1) into XbaI/KpnI digested vector p20-GnTI-CTS-Fc-GFP (Schoberer et al., 2009). The coding DNA sequences for all other chimeric CTS regions were obtained by custom DNA synthesis (GeneArt® Gene Synthesis). The DNA was excised by XbaI/BamHI digestion and ligated into the XbaI/BamHI sites of p20-Fc (expression of GFPglyc tagged proteins), p31 (expression of mRFP tagged proteins), pF (expression of GALT fusions containing the catalytic domain This article is protected by copyright. All rights reserved. Accepted Article of human β1,4-galactosyltransferase), p57 (complementation of gntI plants with the full-length GnTI protein) or p46 (expression of proteins containing a CTS fused to the catalytic domain of tobacco GnTI and GFP). For the generation of pF vector, the human GALT catalytic domain was amplified by PCR using primers GALT18F/19R and the BamHI/XhoI digested PCR product was ligated into the BamHI/SalI sites of pPT2M. For the generation of the pF-GCSI construct the CTS region from A. thaliana αglucosidase I (Saint-Jore-Dupas et al., 2006) was excised by XbaI/BamHI digestion from GCSI-GFPglyc and cloned into pF. In p31, p20-Fc and pF the expression is under the control of the CaMV35S promoter. Vector p57 was generated by insertion of an assembled DNA fragment containing the 386 bp minimal promoter region from A. thaliana GnTI, the A. thaliana GnTI CTS region (AtNNN) and the GnTI catalytic domain into the HindIII/BamHI site of vector p27GFP. For this purpose, the GnTI promoter region was amplified by PCR from genomic DNA using primers AthGnT_12F/13R, the CTS region was amplified with primers AthGnT_14F/16R and the catalytic domain using primers AthGnT_15F/9R. These DNA fragments were assembled using the Gibson Assembly Cloning Kit (NEB). For vector p46 the catalytic domain of N. tabacum GnTI was amplified by PCR from p20-GnTI (Schoberer et al., 2013) using primers NtGnTI_19F/31R. The PCR product was BamHI/BglII digested and cloned into BamHI digested vector p46. In p46, expression of proteins is under the control of the A .thaliana ubiquitin 10 promoter. The construct for expression of the monoclonal antibody (mAb) and for expression of RRR-mRFP (ST-mRFP), RRR-GFP (ST-GFP), NNN-GFPglyc (GnTI-CTS-GFPglyc), NNNmRFP (GnTI-CTS-mRFP), MNS1-GFPglyc, GCSI-GFPglyc, GnTI-mRFP, MNS1-mRFP and AtCASP-mRFP were all available from previous studies (Schoberer et al., 2013; Schoberer et al., 2010; Strasser et al., 2009). LC-ESI-MS analysis Five-week-old N. benthamiana plants were used for Agrobacterium tumefaciens-mediated transient expression of indicated constructs using the agroinfiltration technique as described previously This article is protected by copyright. All rights reserved. Accepted Article (Schoberer et al., 2009). Expressed CTS-GFPglyc chimera or the mAb were purified 48 h after infiltration. 1 g of infiltrated leaves was harvested, homogenized in liquid nitrogen using a mixer mill and resuspended in 600 μL pre-cooled extraction buffer (1 x PBS). After a brief incubation on ice the extract was cleared by centrifugation (9000 g for 20 min at 4°C) and incubated for 1.5 h at 4°C with 20 μL rProteinA Sepharose™ Fast Flow (GE Healthcare). The sepharose was collected by centrifugation, washed three times with 1 x PBS using Micro Bio-Spin™ Chromatography Columns (Bio-Rad) and the bound protein was eluted by incubation in Laemmli buffer for 5 min at 95°C. Approximately 1 µg of chimeric CTS-GFPglyc or mAb was separated by SDS–PAGE (10%) under reducing conditions and stained with Coomassie Brilliant Blue. The corresponding protein band was excised from the gel, destained, carbamidomethylated, in-gel trypsin digested and analyzed by liquidchromatography electrospray ionization-mass spectrometry (LC-ESI-MS) as described in detail previously (Schoberer et al., 2009; Stadlmann et al., 2008). A detailed explanation of N-glycan abbreviations can be found at http://www.proglycan.com. Complementation of A. thaliana gntI plants A. thaliana gntI knockout plants (SALK_073560) (Kang et al., 2008) were transformed with different p57 constructs by floral dipping as described previously (Strasser et al., 2004). Hygromycin-resistant plants were screened by PCR with GnTI and ST-specific primers and selected PCR products were subjected to DNA sequence. Proteins were extracted from leaves of five-week old plants, subjected to SDS-PAGE (10%) under reducing conditions and analyzed by immunoblotting with anti-horseradish peroxidase antibodies (anti-HRP, Sigma) that bind to complex N-glycans carrying β1,2-xylose and core α1,3-fucose residues (Strasser et al., 2004). This article is protected by copyright. All rights reserved. Accepted Article Confocal imaging of fluorescent protein fusions Leaves of five-week-old N. benthamiana plants were infiltrated with agrobacterium suspensions carrying the protein(s) of interest with the following optical densities (OD600): NNN, RRR, NNR, RRN, NRN, RNR, RNN and NRR 0.05, mRFP-AtCASP 0.10, RRR-mRFP 0.07. High-resolution images were acquired 2 and 3 days post infiltration (dpi) on an upright Leica SP5 II confocal microscope using the Leica LAS AF software system. GFP and mRFP were excited with the 488-nm and 561-nm laser line, respectively, and detected at 500-530 nm and 600-630 nm, respectively. Dual-color image acquisition of cells expressing both GFP and mRFP was performed simultaneously. Post-acquisition image processing was performed in Adobe Photoshop CS5. Co-localization analyses of co-expressed fluorescent protein fusions Images of cells expressing the GFPglyc-fused protein of interest together with the cis/medial-Golgi marker mRFP-AtCASP (Osterrieder et al., 2009; Renna et al., 2005) and the non-plant trans-Golgi marker RRR-mRFP (Boevink et al., 1998; Renna et al., 2005) respectively, were acquired 3 dpi without Golgi stack immobilization under non-saturating conditions using zoom factor 5 and a 63x/1.40 NA oil immersion objective for NNN, RRR, NRR, and using zoom factor 6 and a 40x/1.25 NA oil immersion objective for NNR, RNN, RNR. The pinhole was set to 1 airy unit and background noise was reduced by line averaging 8. Only cells with comparable GFP and mRFP fluorescence levels were considered for analysis. Side-on views of dual-labelled Golgi stacks were recorded preferentially as the degree of overlap between two colors appeared clearer. The images obtained were used for co-localization analysis using the Pearson’s correlation coefficient. Calculations were made on 28-38 confocal images per co-expressed combination using the ImageJ (version 1.46m) plugin JACoP (Bolte and Cordelières, 2006). As every image contained 5-10 Golgi bodies, 140-380 Golgi bodies were analyzed for each combination. For a graphical display of the distribution of GFP and mRFP fluorescence intensities across stacks, fluorescence intensity profiles (x-axis: length in µm, y-axis: normalized This article is protected by copyright. All rights reserved. Accepted Article intensity) were generated by drawing a line across dual-labelled Golgi stacks using the “Line Profile” intensity tool of the Leica LAS AF software. Statistical analyses were performed using the Student’s ttest for the comparison of two samples assuming equal variances (Figure S3). FRET-FLIM data acquisition and analysis Infiltrated leaf samples were excised and prior to image acquisition treated for 45 to 60 min with the actin-depolymerizing agent latrunculin B (Calbiochem, stock solution at 1 mM in dimethyl sulphoxide) at a concentration of 25 μM to inhibit Golgi movement (Brandizzi et al., 2002b). 2P-FRETFLIM data capture was performed as described previously (Schoberer et al., 2013; Sparkes et al., 2010) using a two-photon excitation microscope at the Central Laser Facility of the Rutherford Appleton Laboratory. Briefly, a two-photon microscope was constructed around a Nikon TE2000-U inverted microscope using custom-made XY galvanometers (GSI Lumonics). Laser light at a wavelength of 920 ± 5 nm was obtained from a mode-locked titanium sapphire laser (Mira 900F, Coherent Lasers), producing 180-fs pulses at 75 MHz. Two-photon excitation at 920 nm was chosen to allow reduced auto-fluorescence emission from chloroplast and guard cells. The laser beam was focused to a diffraction-limited spot through a VC 60x/1.2 NA water immersion objective (Nikon). Fluorescence emission was collected without descanning, bypassing the scanning system, and passed through a BG39 (Comar) filter to block the near infrared laser light. Line, frame, and pixel clock signals were generated and synchronized with an external fast microchannel plate photomultiplier tube (MCP-PMT, Hamamatsu R3809U) used as the detector. These were linked via a time-correlated single-photon-counting PC module SPC830 (Becker and Hickl) to generate the raw FLIM data. Prior to FLIM data collection, the GFP and mRFP expression levels in the plant specimens within the region of interest were confirmed using a Nikon eC1 confocal microscope with excitation at 488 and 543 nm, respectively. A 633-nm interference filter was used to minimize further the contaminating effect of chlorophyll auto-fluorescence emission that would otherwise obscure the mRFP emission. FLIM This article is protected by copyright. All rights reserved. Accepted Article images were analyzed by obtaining excited-state lifetime values of a single cell. Calculations and image processing were made using the SPC Image analysis software (Becker and Hickl). Lifetime values were collected on a single pixel basis from the center of individual Golgi bodies. Decay curves of a single point highlight an optimal single exponential fit when chi square (χ2) values are 1 (points with χ2 from 0.9 to 1.4 were taken). The collected data values were used to generate histograms depicting the distribution of lifetime values of all data points within the samples. Results are from two to three independent experiments (>150 Golgi stacks from 12-13 cells in total). An observed protein-protein interaction is described by the decrease of the donor fluorescence lifetime (quenching) due to energy transfer to the acceptor (Gadella and Jovin, 1995; Krishnan et al., 2003), which can be calculated by measuring the fluorescence lifetime of the donor in the presence and absence of the acceptor (Bastiaens and Squire, 1999) and can be expressed as a percentage of the donor lifetime, a value referred to as “energy transfer efficiency” (E). The percentage efficiency (E%) can be calculated using equation (1) (1) where τDA and τD are the mean pixel-by-pixel excited-state lifetimes of the donor in the presence and absence of the acceptor determined for each pixel. We have previously shown that a reduction of as little as ~200 ps or 8% in the excited state lifetime of the GFP-labelled protein represents quenching through a protein-protein interaction (Osterrieder et al., 2009; Schoberer et al., 2013; Sparkes et al., 2010; Stubbs et al., 2005; Yadav et al., 2013). Since the instrument response (IR) in our setup is determined to be less than 60 ps there was no need to deconvolute the IR function from the sample data decay curves. Thus lifetime differences of larger than 100 ps can be easily resolved. Co-purification and immunoblotting Co-purification experiments were performed as previously described (Hüttner et al., 2012). Briefly, leaves of five-week-old N. benthamiana plants were co-infiltrated with agrobacteria (OD600 of 0.2) This article is protected by copyright. All rights reserved. Accepted Article containing p20-GnTI-CTS-Fc-GFP (NNN-GFPglyc) and different p31 constructs expressing the chimeric CTS regions (RNR, NRR, RNN, NNR) or controls (NNN, RRR) fused to mRFP. NNN-GFPglyc was purified by binding to rProtein A-Sepharose™ Fast Flow as described in detail recently (Hüttner et al., 2012) and immunoblot detection was performed using anti-GFP (MACS Miltenyi Biotec) and anti-mRFP (ChromoTek) antibodies. Similarly, constructs for expression of chimeric CTS-GFPglyc were coexpressed with NNN-mRFP and analyzed in the same way. For analysis of MNS1-CTS and NNN interaction, MNS1-CTS-GFPglyc was co-expressed with constructs expressing chimeric CTS-regions fused to mRFP and for monitoring of the interaction between full-length proteins NNN-GNTI-GFP or RNR-GNTI-GFP (in p46) were co-expressed with NNN-GNTI-mRFP (in p31), co-purified using GFPTrap-A beads (ChromoTek) and analyzed by immunoblotting. Acknowledgements (25 words) This work was supported by the Austrian Science Fund Project: P23906–B20 and by the Science and Technology Facilities Council Program (access grant to C.H.). A.C. was funded by the Austrian Research Promotion Agency Laura Bassi Centres of Expertise Grant 822757. Supporting Information Table S1. Primers used in this study. Figure S1. LC-ESI-MS spectra of NRN and RRN glycoreporter fusion proteins display also the unglycosylated peptide. Figure S2. 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Yadav, R.B., Burgos, P., Parker, A.W., Iadevaia, V., Proud, C.G., Allen, R.A., O'Connell, J.P., Jeshtadi, A., Stubbs, C.D. and Botchway, S.W. (2013) mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging. BMC Cell Biol, 14, 3. This article is protected by copyright. All rights reserved. Accepted Article Table 1. FRET efficiency determined by FLIM Donor Acceptor NNN-GFPglyc NNN-mRFP NNN-GFPglyc NNN-GFPglyc NNN-GFPglyc NNN-GFPglyc D, RRR-mRFP RNR-mRFP RNN-mRFP NRR-mRFP D ± s.d. (ns) DA 2.44 ± 0.06 2.08 ± 0.09 (n = 412) (n = 204) 2.44 ± 0.06 2.36 ± 0.06 (n = 412) (n = 238) 2.44 ± 0.06 2.26 ± 0.05 (n = 412) (n = 217) 2.44 ± 0.06 2.18 ± 0.07 (n = 412) (n = 185) 2.44 ± 0.06 2.36 ± 0.06 (n = 241) (n = 158) lifetime of the donor in the absence of the acceptor; of the acceptor; Δ , lifetime contrast ( DA]) ± s.d. (ns) D- DA); DA, lifetime Δ (ns) E (%) 0.36 14.56 0.08 3.35 0.18 7.34 0.26 10.79 0.08 3.09 of the donor in the presence E, FRET efficiency calculated according to (1-[ D- × 100; ns, nanosecond; s.d., standard deviation. A minimum decrease of the average excited- state fluorescence lifetime of the donor molecule by 0.20 ns or 8% in the presence of the acceptor molecule was considered relevant to indicate interaction. Protein pairs and respective values indicating interaction are shown in bold. Table 2. Summary of major findings. CTS-region Subcellular GnTI- location1 interaction GnTI-interaction gntI (FRET-FLIM) complementation (Co-IP) This article is protected by copyright. All rights reserved. Accepted Article 1 +++ NNN cis/medial-Golgi +++ +++ RRR trans-Golgi - - RNR cis/medial-Golgi - -/+ ++ NRR trans-Golgi - - - RNN cis/medial-Golgi +++ +++ +++ NNR cis/medial-Golgi - n/d ++ NRN ER n/a n/a n/a RRN ER n/a n/a n/a - Based on data from glycan-analyses as well as from quantification of confocal images n/a: not applicable; n/d: not done Figures Figure. 1. Schematic presentation of protein fusions. (a) The CTS regions of N- acetylglucosaminyltransferase I (GnTI, NNN) and α2,6-sialyltransferase (ST, RRR) and corresponding amino acid sequences are shown. C denotes the short N-terminal cytoplasmic tail; T indicates the transmembrane domain (underlined in the corresponding amino acid sequence) and S depicts the stem region. Domains marked by “N” are from N. tabacum GnTI and “R” indicates domains from ST. (b) Schematic presentation of the chimeric CTS regions derived by exchange of C, T or S regions (NRN, RNR, RNN, NRR, NNR, RRN). (c) Schematic presentation of reporter protein domains that were fused to the CTS regions. “Y” denotes the single N-glycosylation site present in GFPglyc. The conserved Fc domain from human IgG1 is used for affinity purification. GALT CD: harbours the catalytic domain (CD) of human β1,4-galactosyltransferase. AtGnTI CD: harbours the catalytic domain of A. thaliana GnTI. This construct is expressed under the endogenous GnTI promoter from A. thaliana. GnTI CD harbours the catalytic domain of N. tabacum GnTI. This article is protected by copyright. All rights reserved. Accepted Article Figure 2. LC-ESI-MS analysis of glycoreporter fusion proteins reveals differences in subcellular localization. Mass spectra of glycopeptide 1 (EEQYNSTYR) derived from the glycoprotein part of GFPglyc. GCSI, chimeric construct containing the CTS region from the ER-resident A. thaliana αglucosidase I fused to the glycoreporter. Man5 (Man5GlcNAc2) to Man9 (Man9GlcNAc2), oligomannosidic N-glycans, indicative of ER retention; GnGnXF (GlcNAc2XylFucMan3GlcNAc2), MGnXF (GlcNAcXylFucMan3GlcNAc2), GnAXF2 GalGlcNAc2XylFuc2Man3GlcNAc2) complex N-glycans, processed in the Golgi apparatus. The schematic presentation corresponding to the major N-glycan peak is given. The asterisk denotes the presence of an unspecific peak. Figure 3. Co-expression of chimeric-GALT and N-glycan analysis of a glycoprotein reveal distinct sub-Golgi-targeting regions. LC-ESI-MS of a monoclonal antibody (mAb) co-expressed with chimeric CTS regions fused to GALT. Mass spectra of glycopep de 1 (EEQYNSTYR) or
2 (TKPREEQYNSTYR) derived from the Fc region of the mAb are shown. The 2 glycopeptides differ by 482 Da and different ratios of the two glycopeptides are generated during the sample preparation by incomplete digestion with trypsin (Stadlmann et al., 2008). Peaks derived from glycopeptide 1 are marked by asterisks. (a) N-glycan profiles derived by co-expression of NNN-GALT or RRR-GALT. mAb indicates the N-glycan profile in the absence of any GALT enzyme and GCSI shows the profile generated by co-expression of the ER-retained GCSI-GALT. The schematic presentation corresponding to the major N-glycan peak is given. (b) N-glycan profiles derived by co-expression of mAb with chimeric CTS-GALT enzymes. Figure 4. Subcellular localization of fluorescent domain swap constructs. GFPglyc-fused proteins were expressed transiently in tobacco leaf epidermal cells and analyzed by confocal microscopy 3 days post infiltration (dpi). Each confocal image depicts a representative cell expressing the stated GFPglycfusion (green). Bars = 25 µm. This article is protected by copyright. All rights reserved. Accepted Article Figure 5. Co-localization analysis shows changes in intra-Golgi localization of fluorescent domain swap constructs. Fluorescent protein fusions were transiently expressed in tobacco leaf epidermal cells and analyzed by live-cell confocal microscopy (3 dpi) without fixation or inhibition of Golgi stack motility. Confocal images produced in (a) were used for co-localization analyses in (b). (a) Merged confocal images in the left panel show representative cells co-expressing GFPglyc-fused proteins (green) with the reference marker mRFP-AtCASP (magenta), an Arabidopsis cis/medial-Golgi matrix protein, in Golgi stacks of live cells. Co-localization appears in white. The boxed areas are shown as magnifications in the middle panel. The white line drawn across representative Golgi stacks was used to generate fluorescence intensity profiles shown in the right panel that reflect the distribution of the fluorescence intensity of the respective GFP fusion (green) and mRFP-AtCASP (magenta) along the line. Bars = 10 µm. (b) Co-localization analyses of GFPglyc-fused proteins co-expressed with the cis/medial-Golgi marker mRFP-AtCASP and the non-plant trans-Golgi marker RRR-mRFP, respectively, using the Pearson’s correlation coefficient. Figure 6. The stem region of GnTI is mainly responsible for protein-protein interaction. The indicated proteins were transiently co-expressed in N. benthamiana leaves and the GFP-tagged proteins were purified by incubation with Protein A (a-c) or GFP-coupled beads (d). Immunoblot analysis of protein extracts (input = before incubation with beads) and eluted samples (bound = fraction eluted from beads) with anti-GFP and anti-mRFP antibodies. (a) NNN-GFPglyc was precipitated and co-purified chimeric CTS-mRFP was monitored by immunoblotting. (b) Chimeric CTS-GFPglyc was precipitated and co-purified NNN-mRFP was monitored by immunoblotting. (c) MNS1-GFPglyc was precipitated and co-purified chimeric CTS-mRFP was monitored by immunoblotting. (d) RNR-GNTIGFP and NNN-GNTI-GFP were purified by binding to GFP-coupled beads and co-purified NNN-GNTImRFP was analyzed by immunoblotting. This article is protected by copyright. All rights reserved. Accepted Article Figure 7. Complementation of the A. thaliana gntI mutant by expression of chimeric CTS regions fused to the catalytic domain of A. thaliana GnTI. Proteins were extracted from five-week-old soilgrown plants, separated by SDS-PAGE and complex N-glycans were detected by immunoblotting using antibodies directed against β1,2-xylose and α1,3-fucose containing complex N-glycans. (a) Protein extracts from gntI expressing AtNNN-AtGNTI or RRR-AtGNTI. (b) Protein extracts from gntI expressing RNN-AtGNTI, NRR-AtGNTI, RNR-AtGNTI or NNR-AtGNTI. Ponceau S (P.) staining serves as loading controls. This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved. View publication stats