golgi localization of gnti requires a polar amino …...9 the golgi localization of gnti requires a...

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Short title: Plant GnTI localization 1

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Author for contact: Richard Strasser, Department of Applied Genetics and Cell Biology, University 3

of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria, 4

[email protected] 5

phone: ++43-1-47654-94145, fax: ++43-1-47654-94009 6

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The Golgi localization of GnTI requires a polar amino acid residue within its 9

transmembrane domain 10

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Jennifer Schoberer1, Eva Liebminger1, Ulrike Vavra1, Christiane Veit1, Clemens Grünwald-Gruber2 13

Friedrich Altmann2, Stanley W. Botchway3, Richard Strasser1 14

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1Department of Applied Genetics and Cell Biology, University of Natural Resources and Life 16

Sciences, Vienna, Austria 17

2Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria 18

3Research Complex at Harwell, Central Laser Facility, Science and Technology Facilities Council, 19

Rutherford Appleton Laboratory, Harwell-Oxford, Didcot OX11 0QX, United Kingdom 20

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Author for correspondence: [email protected] 22

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Author contributions: J.S. and R.S. conceived the original research plan, designed the experiments 24

and analysed the data; J.S. and E.L. performed most of the experiments; U.V. and C.V. provided 25

technical assistance. C.G-G. and F.A. performed MS-based analysis and interpretation of the data. 26

S.W.B. assisted in design and interpretation of fluorescence imaging data. R.S. wrote the manuscript 27

with contributions from all the authors. 28

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This work was supported by grants from the Austrian Science Fund (FWF): P23906-B20 (to R.S.), 30

P28218-B22 (to R.S.), T655-B20 (to J.S.) and the Mizutani Foundation for Glycosciences (to R.S.). 31

We thank the STFC for funding access to the Central Laser Facility (grant no. 12130006 to Chris 32

Hawes, Oxford Brookes University). 33

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One-sentence summary 35

An amino acid sequence motif in the N-acetylglucosaminyltransferase I transmembrane domain 36

governs its Golgi localization and glycan processing function in plants. 37

Plant Physiology Preview. Published on April 10, 2019, as DOI:10.1104/pp.19.00310

Copyright 2019 by the American Society of Plant Biologists

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ABSTRACT 39

The Golgi apparatus consists of stacked cisternae filled with enzymes that facilitate the sequential and 40

highly controlled modification of glycans from proteins that transit through the organelle. Although 41

the glycan processing pathways have been extensively studied, the underlying mechanisms that 42

concentrate Golgi-resident glycosyltransferases and glycosidases in distinct Golgi compartments are 43

poorly understood. The single-pass transmembrane domain of N-acetylglucosaminyltransferase I 44

(GnTI) accounts for its steady-state distribution in the cis/medial-Golgi. Here, we investigated the 45

contribution of individual amino acid residues within the transmembrane domain of Arabidopsis 46

thaliana and Nicotiana tabacum GnTI towards Golgi localization and N-glycan processing. Conserved 47

sequence motifs within the transmembrane domain were replaced with those from the established 48

trans-Golgi enzyme 2,6-sialyltransferase (ST) and site-directed mutagenesis was used to exchange 49

individual amino acid residues. Subsequent subcellular localization of fluorescent fusion proteins and 50

N-glycan profiling revealed that a conserved glutamine residue in the GnTI transmembrane domain is 51

essential for its cis/medial-Golgi localization. Substitution of the crucial glutamine residue with other 52

amino acids resulted in mislocalization to the vacuole and impaired N-glycan processing in vivo. Our 53

results suggest that sequence-specific features of the GnTI transmembrane domain are required for its 54

interaction with a Golgi-resident adaptor protein or a specific lipid environment that likely promotes 55

COPI-mediated retrograde transport, thus maintaining the steady-state distribution of GnTI in the 56

cis/medial-Golgi of plants. 57

58

INTRODUCTION 59

The Golgi apparatus has a central role in protein processing and transport along the secretory pathway. 60

In the Golgi apparatus, secreted glycoproteins from animals and plants acquire complex glycan 61

structures which furnish them with distinct functions (Helenius and Aebi, 2001; Strasser, 2016). The 62

formation of complex N-glycans in the Golgi is carried out by specific glycosyltransferases and 63

glycosidases that are asymmetrically distributed throughout the different Golgi cisternae (Dunphy and 64

Rothman, 1983; Rabouille et al., 1995). The distinct, yet overlapping, distribution is a prerequisite for 65

controlled glycosylation by spatial separation of glycan processing enzymes that compete for the same 66

acceptor substrates. In mammals, diseases like congenital disorders of glycosylation are caused by 67

missense mutations that affect trafficking and organization of Golgi-resident glycosylation enzymes 68

(Fisher and Ungar, 2016). Despite their important role in maintenance of Golgi organization and 69

function, the mechanisms for steady-state distribution of Golgi-resident glycosyltransferases and 70

glycosidases are still largely unknown (Tu and Banfield, 2010). A better understanding of the Golgi 71

organization is further complicated by the fact that the mode of cargo transport through the Golgi is 72

still controversial in most organisms and different cell types (Glick and Luini, 2011; Ito et al., 2014). 73



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Sequence motifs that govern the intra-Golgi distribution can either function as a retention signal for a 74

distinct Golgi sub-compartment, a retrieval signal for retrograde transport, or a combination of both. 75

A common feature of Golgi-resident N-glycan processing enzymes is their type II membrane protein 76

topology with a short cytoplasmic tail, a single transmembrane domain (TMD) and a stem or linker 77

region that orients the large catalytic domain into the Golgi lumen. Proposed models for Golgi 78

retention and retrieval include oligomerization of glycosyltransferases and glycosidases with 79

subsequent exclusion of large protein complexes from intra-Golgi transport (Machamer, 1991; Nilsson 80

et al., 1993), TMD length-dependent sorting (Munro, 1995), lipid-based partitioning (Patterson et al., 81

2008) and cytoplasmic tail-dependent processes (Uliana et al., 2006; Petrosyan et al., 2015). An 82

adaptor-mediated sorting mechanism involving binding to a specific amino acid motif within the 83

cytoplasmic tail of glycosyltransferases and interaction with the coat protein complex I (COPI) 84

retrograde transport system has been described in yeast and mammals (Schmitz et al., 2008; Tu et al., 85

2008; Eckert et al., 2014). Yeast VPS74 acts as an adaptor protein that binds to the sequence motif in 86

the cytoplasmic tail of late-Golgi glycosyltransferases and to COPI subunits to mediate the retrograde 87

transport in the Golgi. Apart from VPS74 and its mammalian homolog GOLPH3, which may have a 88

similar function, no adaptor proteins for binding to cytoplasmic tails have been identified so far and 89

for many Golgi-resident enzymes, like those involved in complex N-glycan processing in mammals or 90

plants, the signals and mechanisms for their steady-state distribution are not well known. Noteworthy, 91

an adapter-mediated mechanism has not been described in plants and VPS74/GOLPH3-like proteins 92

appear absent. Interestingly, a recent study showed a direct binding of COPI subunits to an amino acid 93

motif present in the cytoplasmic tail of several mammalian glycosyltransferases (Liu et al., 2018) 94

indicating an adaptor-protein independent sorting. Golgi localization of plant N-glycan processing 95

enzymes is typically mediated by sequence features within the N-terminal region comprising the 96

cytoplasmic tail, the TMD and the stem (CTS region) without any contribution from the catalytic 97

domain (Saint-Jore-Dupas et al., 2006; Schoberer et al., 2010; Schoberer et al., 2013). However, 98

within this N-terminal targeting region, a conserved sequence motif contributing to Golgi 99

compartment-specific localization has yet to be revealed. 100

N-glycosylation is an essential posttranslational modification that is initiated in the endoplasmic 101

reticulum (ER) by transfer of a preassembled oligosaccharide to asparagine residues within the 102

consensus sequence Asn-X-Ser/Thr (X can be any amino acid except proline) on nascent proteins 103

(Strasser, 2016). Upon transfer, the N-glycans are processed by specific -glucosidases and -104

mannosidases, and glycoproteins with oligomannosidic N-glycans exit the ER towards the Golgi 105

complex. In the Golgi, additional mannose residues are removed by Golgi -mannosidase I 106

(Liebminger et al., 2009) and complex N-glycan formation is initiated by 1,2-N-107

acetylglucosaminyltransferase I (GnTI, called MGAT1 in mammals), which transfers a single GlcNAc 108

residue to Man5GlcNAc2. This is a critical step in the pathway because all further N-glycan processing 109

reactions in the Golgi depend on the presence of the single GlcNAc added by GnTI (von Schaewen et 110



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al., 1993; Strasser et al., 1999). Importantly, the central function of GnTI in complex N-glycan 111

formation is conserved in higher eukaryotes and GnTI deficiency is embryo-lethal in mammals (Ioffe 112

and Stanley, 1994). GnTI-deficient Lotus japonicus display severe growth defects and Oryza sativa 113

(rice) lacking GnTI show developmental phenotypes leading to early lethality (Fanata et al., 2013; 114

Pedersen et al., 2017). Arabidopsis thaliana lacking GnTI are viable, but less tolerant to abiotic stress 115

and display severe growth defects when combined with mutations that affect in plant cell wall 116

formation (von Schaewen et al., 1993; Kang et al., 2008). In summary, these data highlight the 117

importance of complex N-glycan initiation by GnTI for different biological processes across 118

kingdoms. 119

In accordance with its N-glycan processing function, GnTI is mainly located in the cis/medial-Golgi in 120

plants (Reichardt et al., 2007; Schoberer et al., 2010). In a previous study, we generated chimeric 121

GnTI variants and analyzed their Golgi localization and functionality to identify regions for Golgi 122

targeting and retrieval/retention (Schoberer et al., 2014). The results for Nicotiana tabacum (tobacco) 123

GnTI demonstrated that the cytoplasmic tail, the stem region and the catalytic domain are not involved 124

the intra-Golgi distribution of GnTI. By contrast, the TMD plays a pivotal role for its cis/medial-Golgi 125

distribution. Here, we investigated the contribution of individual sequence motifs and amino acid 126

residues in the TMD to Golgi localization and GnTI function. Tobacco and Arabidopsis GnTI (herein 127

NtGnTI and AtGnTI, respectively) were analyzed to identify a common mechanism for their Golgi 128

distribution. We identified a single amino acid residue conserved in the TMD of plant GnTI enzymes 129

that plays an essential role in GnTI cis/medial-Golgi localization and N-glycan processing. 130

Mutagenesis of this conserved residue altered the subcellular localization of GnTI and impaired its 131

function when expressed under native-like conditions. In summary, our study reveals a motif that 132

governs Golgi-resident enzyme distribution and provides insights into the subcellular localization 133

mechanisms of Golgi-located type II membrane proteins. 134

135

RESULTS 136

The exoplasmic half of the GnTI TMD is important for the cis/medial-Golgi localization 137

We compared the GnTI TMD sequences from different plant species to identify amino acid motifs that 138

are involved in the cis/medial-Golgi distribution. The sequence alignment revealed the presence of 139

several highly conserved amino acid residues, especially in the exoplasmic half of the TMD (Fig. 1A 140

and Suppl. Fig. S1). Consequently, we exchanged the exoplasmic half of the TMD from the well-141

characterized trans-Golgi marker rat 2,6-sialyltransferase (ST) (Boevink et al., 1998) with 9 142

exoplasmic amino acids from the 18-residue TMD of NtGnTI. The resulting chimeric CTS regions, 143

ST/GnTI and GnTI/ST (Fig. 1B and Suppl. Fig. S2), were C-terminally fused to the NtGnTI catalytic 144

domain and GFP, transiently expressed in Nicotiana benthamiana leaves and examined using a 145

confocal microscope. Co-localization was carried out with the 1,2-galactosyltransferase MUR3, a 146

xyloglucan synthesizing enzyme that has been associated with medial-Golgi cisternae (Chevalier et al., 147



5

2010). As expected, both constructs harbouring the chimeric CTS regions displayed Golgi localization 148

(Suppl. Fig. S3). To assess the degree of co-localization in the Golgi, the Pearson’s correlation co-149

efficient was determined for co-localization with the cis-Golgi protein AtCASP (Osterrieder et al., 150

2010; Schoberer et al., 2010) fused to mRFP and the trans-Golgi marker ST-CTS-mRFP (Fig. 1C and 151

Suppl. Table S1). The experiment was carried with the CTS regions lacking the catalytic domains to 152

enhance their expression. The co-localization analysis revealed a similar correlation for ST/GnTI-153

CTS-GFP and NtGnTI-CTS-GFP as well as for GnTI/ST-CTS-GFP and ST-CTS-GFP, indicating that 154

the 9 exoplasmic amino acids from the GnTI TMD contain a sequence motif for the concentration in a 155

distinct Golgi sub-compartment. 156

To obtain further support for our finding, we fused the ST/GnTI-CTS and GnTI/ST-CTS regions to the 157

catalytic domain of human 1,4-galactosyltransferase (GALT) and performed an N-glycan processing 158

assay. In this assay, the co-expression of GALT with a secreted glycoprotein reporter results in the 159

transfer of 1,4-galactose to the non-reducing end of N-glycans on the reporter (Schoberer et al., 160

2014). Dependent on the Golgi compartmental localization of the chimeric GALT, a different N-161

glycan pattern is obtained due to competition of GALT with endogenous N-glycan processing 162

enzymes (Suppl. Fig. S4). Thus, the N-glycan processing assay allows cis/medial-Golgi-localized 163

GALT (leading to incompletely processed N-glycans) to be distinguished from medial/trans-Golgi-164

localized GALT (higher amount of completely processed N-glycans). Mass spectrometry-based 165

analysis of a glycopeptide derived from the reporter revealed incompletely processed N-glycans for 166

ST/GnTI-CTS and a substantial amount of completely processed N-glycans for GnTI/ST-CTS (Suppl. 167

Fig. S5), providing additional evidence for a role of the exoplasmic TMD half in intra-Golgi 168

localization. 169

We have previously shown that a trans-Golgi targeted GnTI carrying the ST-CTS region fails to 170

complement the complex N-glycan processing defect of GnTI-deficient Arabidopsis plants (Schoberer 171

et al., 2014). Here, we fused the chimeric CTS regions with the exchanged exoplasmic TMD domains 172

to the AtGnTI catalytic domain. GFP-fused AtGnTI variants were expressed in Arabidopsis gntI T-173

DNA insertional mutants under the control of a weak endogenous AtGnTI promoter and the processing 174

to complex N-glycans was analyzed by immunoblotting. Consistent with an effect on cis/medial-Golgi 175

localization, GnTI/ST-AtGnTI-GFP carrying the exoplasmic TMD region from ST did not restore 176

complex N-glycan formation (Suppl. Fig. S6). However, 8 out of 12 tested transgenic ST/GnTI-177

AtGnTI-GFP lines displayed considerable amounts of complex N-glycans indicating relocation of the 178

protein to an earlier Golgi compartment where it initiated complex N-glycan processing. Taken 179

together, our results show that an amino acid motif in the exoplasmic half of the NtGnTI TMD 180

contains a signal for efficient steady-state cis/medial-Golgi localization. 181

182

A single amino acid residue in the NtGnTI TMD is crucial for cis/medial-Golgi localization 183



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To further characterize the sequence motif in the exoplasmic half of the NtGnTI TMD, we generated 184

another chimeric GALT variant where the LFAV tetrapeptide sequence from ST was replaced by the 185

FIYIQ amino acid motif from NtGnTI (STFIYIQ-GALT) and carried out the N-glycan processing assay. 186

Co-expression of STFIYIQ-GALT resulted in the generation of incompletely processed N-glycans 187

(Suppl. Fig. S5), indicating that the FIYIQ-motif plays a role in NtGnTI cis/medial-Golgi localization. 188

One of the two fully conserved residues in this motif is the glutamine at position 25 (Fig. 1A). We 189

replaced this glutamine (Q25) with alanine and analyzed the localization of NtGnTI-Q25A fused to 190

GFP or mRFP in N. benthamiana leaf epidermal cells. NtGnTI-Q25A was found in Golgi bodies as 191

well as other intracellular compartments indicating that the subcellular localization was changed 192

(Suppl. Fig. S3). Co-localization of NtGnTI-Q25A-GFP with mRFP-AtCASP and ST-CTS-mRFP 193

indicated a shift in the Golgi localization towards the trans-Golgi (Fig. 1D). The N-glycan processing 194

assay revealed the presence of processed N-glycans when NtGnTI-Q25A-GALT was co-expressed 195

with the glycoprotein reporter and the N-glycan profile was comparable to that derived from ST-196

GALT (Fig. 1E). 197

In addition to the accumulation in the trans-Golgi, GFP- or mRFP-tagged NtGnTI-Q25A was 198

observed in the vacuole (Fig. 2A and Suppl. Fig. S3), which was clearly visible three to four days after 199

infiltration. Co-localization with aleu-GFP (Humair et al., 2001) confirmed targeting to the lytic 200

vacuole (Fig. 2B). No ER localization was observed for NtGnTI-Q25A fusion proteins, but 201

accumulation of fluorescence in the apoplast was occasionally observed indicating the secretion of 202

full-length NtGnTI-Q25A or a degradation product. In addition to vacuolar and trans-Golgi 203

localization, we observed smaller puncta in NtGnTI-Q25A expressing cells that did not represent 204

typical Golgi bodies (Fig. 2C). Additional co-localization experiments with different subcellular 205

markers were carried out to identify these small puncta. However, the cellular structures overlapped 206

with neither the TGN marker mRFP-SYP61 (Sanderfoot et al., 2001) (Fig. 2D), the early endosome 207

marker GFP-RABF2b (ARA7) (Kotzer et al., 2004) (Fig. 2E), the autophagosome marker GFP-208

ATG8e (Contento et al., 2005) (Fig. 2F), nor the late endosome/multivesicular body marker GFP-209

RABF1 (ARA6) (Ueda et al., 2001) (Fig. 2G). These data show that the glutamine residue confers 210

efficient cis/medial-Golgi localization of NtGnTI. The mutated NtGnTI-Q25A, on the other hand, 211

accumulates in the trans-Golgi and substantial amounts are released to post-Golgi compartments 212

including the vacuole by an unknown trafficking route. 213

214

Arabidopsis GnTI-Q23A fails to complement GnTI-deficient plants 215

To investigate the functional relevance of the identified glutamine residue within the TMD of GnTI, 216

we expressed GFP- and mRFP-tagged AtGnTI and the AtGnTI-Q23A variant with the conserved 217

glutamine (at position 23 in AtGnTI) changed to an alanine in the gntI mutant. A functional AtGnTI in 218

the cis/medial-Golgi will restore the N-glycan processing which can be analyzed by immunoblotting 219

using antibodies directed against Golgi-processed N-glycans carrying 1,2-xylose and core 1,3-220



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fucose (Strasser et al., 2004). When expressed under the control of the weak AtGnTI promoter, 221

complex N-glycans were detected in gntI plants expressing AtGnTI-GFP, but not in AtGnTI-Q23A-222

GFP expressing plants (Fig. 3A). AtGnTI-GFP could be found on immunoblots, whereas AtGnTI-223

Q23A-GFP was hardly detectable. Comparison of the transcript levels between the two AtGnTI 224

variants by reverse transcription quantitative PCR (RT-qPCR) did not reveal considerable variation in 225

mRNA levels (Fig. 3B), suggesting that the difference in protein abundance is caused by a decreased 226

stability of the AtGnTI-Q23A-GFP protein. The same result was obtained for complementation of 227

Arabidopsis cgl1 plants that carry a missense mutation in the AtGnTI gene (von Schaewen et al., 1993; 228

Strasser et al., 2005) or when mRFP-tagged AtGnTI variants were used for complementation of gntI 229

(Suppl. Fig. S7). MALDI-TOF MS analysis of N-glycans from gntI expressing AtGnTI-GFP under the 230

control of the weak endogenous AtGnTI promoter showed partial formation of complex N-glycans. By 231

contrast, no peaks corresponding to complex N-glycans were detectable for AtGnTI-Q23A-GFP 232

(Suppl. Fig. S8). Expression of AtGnTI-mRFP or AtGnTI-Q23A-mRFP under the control of the 233

constitutive ubiquitin 10 (UBQ10) promoter restored the complex N-glycan formation in gntI plants 234

(Fig. 3C). However, in gntI expressing the mutant AtGnTI-Q23A variant, MALDI-TOF MS analysis 235

revealed the presence of considerable amounts of unprocessed Man5GlcNAc2 (Man5), which is the 236

major N-glycan in AtGnTI-deficient plants (Fig. 3D and Suppl. Fig. S8) (von Schaewen et al., 1993; 237

Strasser et al., 2005). Thus, even the overexpression of the AtGnTI-Q23A variant could not fully 238

complement the N-glycan processing defect of gntI which would result in reduced levels of Man5 and 239

more of the truncated N-glycan carrying 1,2-xylose and core 1,3-fucose (MMXF). This finding is 240

fully consistent with the observed mistargeting due to the disturbance of the conserved sequence motif 241

within the GnTI TMD. 242

Immunoblot analysis of AtGnTI-mRFP stably expressed under the control of the UBQ10 promoter in 243

gntI revealed a single band of the expected size of approximately 77 kDa for the fusion protein. By 244

contrast, the AtGnTI-Q23A variant displayed 3 to 4 bands (Fig. 3C). The upper one corresponds to the 245

size of the full-length AtGnTI-mRFP protein, whereas the other 2–3 bands represent faster migrating 246

variants of different sizes suggesting the presence of degradation products. In a previous study, we 247

observed that GnTI forms a homodimer which requires a sequence domain from the stem region 248

(Schoberer et al., 2013; Schoberer et al., 2014). Here, we tested whether mistargeted AtGnTI-Q23A 249

can still interact with the non-mutated AtGnTI. Both proteins were co-expressed in N. benthamiana 250

leaf epidermal cells and subjected to co-immunoprecipitation analysis. In contrast to wild-type 251

AtGnTI, a substantially decreased amount of AtGnTI-Q23A was co-purified (Fig. 3E). Notably, only 252

the band representing full-length AtGnTI interacted, whereas the faster migrating bands from AtGnTI-253

Q23A were not co-purified providing potential evidence that they represent spatially separated 254

variants from post-Golgi compartments. FRET-FLIM analysis of transiently expressed NtGnTI and 255

NtGnTI-Q25A fusion proteins showed that the FRET efficiency determined by FLIM was also 256

reduced for the wild-type/mutant NtGnTI combination compared to the homodimer formation of wild-257



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type NtGnTI (Table 1). Confocal microscopy of transgenic seedlings confirmed the Golgi localization 258

of AtGnTI-mRFP. AtGnTI-Q23A-mRFP-expressing seedlings hardly displayed any Golgi 259

localization, but showed a fluorescence signal in the apoplast which may be derived from the cleavage 260

and secretion of mRFP. A similar secretion has been described for the trans-Golgi marker ST-mRFP 261

(Runions et al., 2006), but in the case of AtGnTI-Q23A-mRFP, the effect is less pronounced (Fig. 3F). 262

To investigate the fate of the mutant variant in more detail, 10-day-old seedlings were treated with 263

cycloheximide and the AtGnTI-mRFP protein level was analyzed at different time points by 264

immunoblotting. AtGnTI-mRFP levels were stable during the chase period. The AtGnTI-Q23A-mRFP 265

levels, on the other hand, decreased over time indicating a faster clearance (Fig. 4A). Moreover, we 266

treated Arabidopsis seedlings with the proteasomal inhibitor MG132 (Fig. 4B) as well as with the 267

vacuolar H+-ATPase inhibitor concanamycin A (Dettmer et al., 2006) to see whether the discrete 268

degradation products were generated in an acidic compartment like the vacuole (Fig. 4C). By 269

increasing the pH in the organelle, the catalytic activity of proteases is diminished which may result in 270

the accumulation of proteins targeted for vacuolar degradation (Chamberlain et al., 2008). 271

Pharmacological inhibition with MG132 or concanamycin A did not result in substantial changes in 272

the pattern of AtGnTI-Q23A-mRFP degradation products. However, incubation of Arabidopsis 273

seedlings in a combination of MG132 and concanamycin A increased the amounts of the intact 274

AtGnTI-Q23A-mRFP protein (Fig. 4D). By contrast, the ER-associated degradation inhibitor 275

kifunensine did not stabilize AtGnTI-Q23A-RFP (Fig. 4E). 276

277

A polar or positively charged amino acid is required for proper Golgi targeting and in vivo N-278

glycan processing 279

Different features of the TMD have been suggested to play a role for the subcellular localization of 280

type II membrane proteins including the TMD length, the presence of polar residues in the exoplasmic 281

region of the leaflet or changes in the average amino acid volume in the exoplasmic half (Munro, 282

1995; Sharpe et al., 2010; Quiroga et al., 2013). To define the signal for proper AtGnTI localization in 283

more detail, we analyzed the contribution of different amino acid residues at this position to GnTI 284

localization and processing of complex N-glycans. We performed site-directed mutagenesis and 285

generated mutants where Q23 from AtGnTI was replaced by His, Leu, Glu, Tyr, Val and Ser. 286

Confocal microscopy of mRFP-fusion proteins expressed under the control of the ubiquitin 10 287

promoter showed that AtGnTI-Q23V, AtGnTI-Q23L and AtGnTI-Q23S behaved like AtGnTI-Q23A 288

and displayed mistargeting to post-Golgi compartments. AtGnTI-Q23H showed mainly Golgi 289

localization similar to that of AtGnTI. AtGnTI-Q23E and AtGnTI-Q23Y displayed Golgi localization 290

as well as post-Golgi distribution (Fig. 5A and Suppl. Fig. S9). In agreement with altered localization, 291

an increase of the faster migrating degradation product was observed for various AtGnTI mutants (Fig. 292

5B). Together, these data suggest that a polar amino acid residue with a long side chain (glutamine) or 293

a positively charged residue like histidine is tolerated at this particular position within the GnTI TMD, 294



9

whereas hydrophobic residues with small (e.g. alanine) or large volumes (e.g. leucine) are not 295

tolerated (Suppl. Fig. S10). To determine the effect of the different residues on in vivo N-glycan 296

processing, transgenic Arabidopsis gntI expressing the different mutant variants under the control of 297

the AtGnTI promoter were generated. In agreement with our localization data, only AtGnTI-Q23H-298

RFP displayed a similar degree of complex N-glycan formation like AtGnTI-RFP. Other mutants did 299

not complement the defect at all or resulted only in partial complementation (e.g. AtGnTI-Q23E) (Fig. 300

5C). Likewise, substitution of adjacent highly conserved amino acids (F19A, M24I and R25A) did not 301

obviously affect the sub-Golgi distribution of AtGnTI and resulted in gntI complementation (Suppl. 302

Fig. S11). 303

304

The Arabidopsis Golgi -mannosidase MNS1 is mistargeted and non-functional when fused to 305

the AtGnTI-CTS-Q23A domain 306

The conserved glutamine in the TMD is important for the cis/medial-Golgi localization of GnTI. To 307

see whether other N-glycan processing enzymes are mistargeted when their CTS region is exchanged 308

with the mutated GnTI domain, we fused the AtGnTI CTS region to the catalytic domain of the 309

Arabidopsis Golgi -mannosidase MNS1, which trims mannose residues from N-glycans in the Golgi 310

and acts upstream of GnTI by converting Man8GlcNAc2 to Man5GlcNAc2 (Liebminger et al., 2009). 311

We generated chimeric AtGnTI-CTS-MNS1-GFP and AtGnTI-CTS-Q23A-MNS1-GFP constructs, 312

where the CTS region from GnTI is fused to the catalytic domain of MNS1 and GFP. The chimeric 313

proteins were expressed under the control of the endogenous MNS1 promoter in the mns1 mns2 mns3 314

triple mutant that generates mainly Man9GlcNAc2 structures. Restoration of MNS1 activity in the 315

cis/medial-Golgi of mns1 mns2 mns3 leads to the formation of complex N-glycans (Liebminger et al., 316

2009). In contrast to AtGnTI-CTS-MNS1-GFP, the MNS1 variant with the Q23A substitution in the 317

AtGnTI TMD failed to restore complex N-glycan formation (Fig. 6A) and no Golgi localization was 318

detected in root cells (Fig. 6B). We previously showed that the mns1 mns2 mns3 mutant displays a 319

severe root-growth phenotype due to the block in N-glycan processing (Liebminger et al., 2009). 320

Expression of MNS1:AtGnTI-CTS-MNS1-GFP in mns1 mns2 mns3 led to an almost wild type-like 321

root growth of seedlings whereas the MNS1 variant harbouring the GnTI targeting region with the 322

mutated glutamine displayed aberrantly swollen and truncated roots (Fig. 6C). The difference was 323

even more pronounced when seeds were grown on 4% sucrose which enhances the phenotype (Fig. 6D 324

and Suppl. Fig. S12). These results highlight the importance of proper Golgi localization mediated by 325

the glutamine-containing sequence motif in the GnTI TMD. 326

327

Knockdown of COPI subunits leads to GnTI mislocalization to the vacuole 328

To investigate whether COPI subunits are involved in Golgi localization of GnTI, we carried out 329

immunoprecipitation of AtGnTI-mRFP and AtGnTI-Q23A-mRFP and monitored if endogenous 330

Arabidopsis COP is co-purified. Immunoblot analysis did not reveal any signal corresponding to 331



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COP suggesting that there is no or only a very weak interaction with AtGnTI (Suppl. Fig. S13). Next, 332

we generated RNAi constructs to silence endogenous COP and COP in N. benthamiana leaves. 333

When the RNAi constructs were transiently co-expressed with the Golgi protein RFP-EMP12 that 334

carries a COPI binding site, mistargeting to the vacuole was observed which is in agreement with 335

earlier studies (Gao et al., 2014; Woo et al., 2015). Likewise, NtGnTI-mRFP was mislocalized to the 336

vacuole in cells co-infiltrated with COP or COP RNAi constructs (Fig. 7A and Suppl. Fig. S13) and 337

the trans-Golgi-resident ST-NtGnTI-mRFP was found extracellularly, as described previously (Ahn et 338

al., 2015), and in the vacuole (Fig. 7A). Silencing of these COPI subunits targeted AtGnTI-mRFP (or 339

AtGnTI-GFP) to the vacuole, whereas for AtGnTI-mRFP-Q23A, the vacuolar localization was not 340

changed. AtGnTI-mRFP-Q23A was also localized predominately to the vacuole when incubated for 1 341

or 2 h in brefeldin A (BFA) (Fig. 7B). Immunoblot analysis revealed an accumulation of the intact 342

mRFP fusion protein upon co-expression of the COPI silencing constructs accompanied by an increase 343

of cleaved mRFP (Fig. 7C). This finding indicates that the Golgi-resident GnTI is not efficiently 344

recycled in the Golgi complex when COPI formation is impaired leading to vacuolar targeting. 345

Moreover, transient overexpression of AtGnTI-mRFP in N. benthamiana caused an increase in 346

truncated fragments (Suppl. Fig. S13), suggesting a saturation of the GnTI localization mechanism. 347

348

DISCUSSION 349

How can a distinct amino acid motif within the transmembrane domain mediate specific 350

subcellular localization of a Golgi-resident protein? 351

A large number of secretory proteins are glycosylated and undergo glycan processing reactions in the 352

Golgi. The concentration of processing enzymes in distinct Golgi cisternae is a prerequisite for the 353

sequential and orderly modification of glycans. However, the organization of Golgi-resident enzymes 354

and the mechanism of cargo transport through the Golgi are under debate. In the stable 355

compartment/vesicular transport model, the cargo processing enzymes remain in their cisternae and 356

cargo is transported from the cis-Golgi to the trans-Golgi by vesicular transport and/or transient 357

tubular connections (Rabouille and Klumperman, 2005; Glick and Luini, 2011). An important feature 358

of the model is the absence of Golgi-resident enzymes in anterograde trafficking carriers and the 359

differential localization of Golgi-resident enzymes is achieved by retention in specific Golgi cisternae. 360

According to this model, the motif in the GnTI TMD constitutes a cis/medial-Golgi retention signal 361

that prevents anterograde transport. 362

In the cisternal maturation model, cargo stays in the cisternae, which matures from cis-to-trans 363

together with Golgi-resident enzymes. To maintain their gradient-like distribution across the Golgi 364

stack, the cargo processing enzymes are cycled back from late to earlier Golgi cisternae by COPI 365

vesicles (Villeneuve et al., 2017). The recycling of Golgi processing enzymes transforms 366

biosynthetically inactive cisternae into biosynthetically active cisternae (Donohoe et al., 2013). In the 367

cisternal maturation model, the GnTI TMD motif acts as a signal for incorporation into retrograde 368



11

transport carriers that mediate recycling of the glycosyltransferase. Even though an interaction with 369

COPI subunits was not found so far, the mislocalization of GnTI to post Golgi compartments after 370

COP and COP knockdown can be explained by impaired recycling from the trans-Golgi to an 371

earlier cisternae as proposed by the cisternal maturation model (Fig. 8). The existence of two types of 372

COPI vesicles in plants has been shown by electron tomography (Donohoe et al., 2007). COPIa-type 373

vesicles are located in the vicinity of cis cisternae and likely contribute to Golgi-to-ER transport. 374

COPIb-type vesicles have been found next to medial- and trans-Golgi cisternae. Immunolabelling 375

showed that the cis/medial-Golgi N-glycan processing enzyme MNS1 that generates the N-glycan 376

substrate for GnTI is present in COPIb-type vesicles (Donohoe et al., 2007). Likewise, GnTI may also 377

be incorporated into COPIb-type carriers that cycle from the trans-Golgi to the cis/medial-Golgi. In 378

the absence of recycling, GnTI accumulates in the trans-Golgi from where it is transported to post-379

Golgi compartments and the vacuole. A comparable recycling mechanism has been proposed for the 380

cis/medial-Golgi-located multiple transmembrane protein AtEMP12 (Gao et al., 2012; Woo et al., 381

2015). AtEMP12 has a conserved COPI-interacting KXD/E motif in its C-terminal cytoplasmic 382

region. This amino acid sequence is not present in the short cytoplasmic tail of GnTI and the 383

conserved glutamine residue in the exoplasmic half of the transmembrane protein is not accessible for 384

a direct interaction with COPI subunits. Therefore, GnTI may bind via its transmembrane domain to 385

an integral membrane protein which serves as an adaptor for incorporation into COPI carriers. 386

The polytopic yeast membrane proteins ERV14 and RER1 are cargo sorting receptors that recognize 387

transmembrane domain features and sort cargo into COPII or COPI carriers (Sato et al., 2001; Herzig 388

et al., 2012; Gomez-Navarro and Miller, 2016). Interestingly, RER1 recognizes polar residues in the 389

transmembrane domain of single TMD proteins such as SEC12 or SEC71 (Sato et al., 2001, 2003) and 390

retrieves them from the Golgi to the ER. Moreover, proper localization of the yeast type II membrane 391

protein ER 1,2-mannosidase I depends on the presence of RER1 (Massaad et al., 1999). The TMD of 392

ER 1,2-mannosidase I interacts with RER1 and ER 1,2-mannosidase I is missorted to the vacuole in 393

the absence of a functional RER1. The Arabidopsis RER1 family consists of three members that can 394

complement the mislocalization of cargo proteins in Saccharomyces cerevisiae rer1, thus suggesting 395

a similar function in plants. However, RER1-interacting proteins and a role of RER1 in intra-Golgi 396

transport have not been described in plants. In addition to ERV14 and RER1, EMP family proteins are 397

another group of Golgi-resident integral membrane proteins that may directly interact with GnTI and 398

play a role in its subcellular localization. 399

Apart from a direct protein-protein interaction and the involvement of an adapter protein, the motif in 400

the GnTI TMD may be crucial for a specific protein-lipid interaction to partition GnTI into a 401

membrane domain competent for recycling to earlier Golgi cisternae (Fig. 8). In mammalian cells, the 402

interaction of a distinct sequence motif in the transmembrane domain of p24 with an individual 403

sphingolipid affects the COPI-dependent retrograde transport (Contreras et al., 2012). Moreover, 404

disruption of sphingomyelin homeostasis has led to changes in glycan processing by disturbance of 405



12

glycosyltransferase organization in distinct Golgi membranes (van Galen et al., 2014). In plants, 406

alterations in the Golgi lipid composition affect protein secretion and Golgi morphology, but the Golgi 407

localization of the trans-Golgi marker ST appears unaltered (Melser et al., 2010). 408

The function of GnTI in N-glycan processing as well as its subcellular localization are conserved in 409

mammals and plants (von Schaewen et al., 1993; Gomez and Chrispeels, 1994; Bakker et al., 1999; 410

Strasser et al., 1999) and there is evidence for cycling of mammalian GnTI (Hoe et al., 1995). A 411

recently identified COPI-binding motif in mammalian glycosyltransferases is not present in the 412

cytoplasmic tail of human GnTI and, in contrast to other cis/medial-Golgi enzymes, no direct binding 413

of GnTI to COPI subunits was found (Liu et al., 2018). GOLPH3, the mammalian VPS74 homolog, is 414

involved in the incorporation of certain glycosyltransferases into COPI vesicles (Eckert et al., 2014) 415

but its absence had no effect on GnTI localization in human cells (Liu et al., 2018). A role for the 416

TMD of mammalian GnTI in Golgi localization has also been described in other studies (Burke et al., 417

1992; Tang et al., 1992; Burke et al.; Nilsson et al., 1996) and the TMD from mammalian GnTIs share 418

a sequence motif with a highly conserved asparagine residue at a similar position. Whether this residue 419

or other features of the TMD are involved in intra-Golgi localization of mammalian GnTI remains to 420

be shown. A general role of the TMD in Golgi retention of type II membrane proteins has been 421

proposed for yeast and mammals (Quiroga et al., 2013) and individual amino acids including polar 422

residues within the TMD contribute to Golgi localization of mammalian proteins (Aoki et al., 1992; 423

Machamer et al., 1993), but a specific receptor or lipid interaction has not been described. 424

In summary, our data show that a transmembrane motif-based sorting mechanism mediates the steady-425

state localization of GnTI in plants. Our findings are fully consistent with the fact that GnTI homomer 426

formation plays no or only a minor role in cis/medial Golgi localization (Schoberer et al., 2013; 427

Schoberer et al., 2014). The mechanism which is functional in Arabidopsis as well as in N. 428

benthamiana is distinct from the earlier proposed bilayer thickness-mediated retention, the kin 429

recognition and the more recently recognized cytoplasmic tail-dependent sorting (Tu and Banfield, 430

2010). Our model proposes that a conserved polar amino acid in the TMD promotes the interaction 431

with a yet unknown adaptor protein/complex or the segregation into a distinct lipid domain. Impaired 432

recycling in the absence of the specific interaction results in GnTI exit from the Golgi and trafficking 433

to post-Golgi compartments including the vacuole where GnTI is eventually degraded. This trafficking 434

route may highlight the default degradation pathway for glycosyltransferases that are not needed 435

anymore in a plant cell. 436

437

MATERIALS AND METHODS 438

Plant material and growth conditions 439

Arabidopsis thaliana (ecotype Col-0) and mutant plants were grown at 22°C under long-day 440

conditions (16-h-light/8-h-dark photoperiod) on soil or on 0.5 × Murashige and Skoog (MS) medium 441

containing 1% (w/v) sucrose and 0.8% (w/v) agar. The mns1 mns2 mns3 (Liebminger et al., 2009), 442



13

gntI (Schoberer et al., 2014) and cgl1 (von Schaewen et al., 1993; Strasser et al. 2005) mutants were 443

available from previous studies. For the measurement of the root length, Arabidopsis seedlings were 444

grown for 10 days on 0.5 x MS medium supplemented with 4% sucrose. For the inhibitor treatments, 445

Arabidopsis seedlings were incubated for 17 h in liquid MS medium supplemented with 1 µM 446

concanamycin A (Sigma Aldrich), 20 µM MG132 (Sigma Aldrich), 100 µg/ml cycloheximide (Sigma 447

Aldrich) or 20 µM kifunensine (Santa Cruz Biotechnology). Nicotiana benthamiana were grown 448

under long-day conditions at 24°C. 449

450

Cloning of constructs 451

DNA fragments coding for the chimeric GnTI/ST-, ST/GnTI- and STFIYIQ-CTS regions were obtained 452

by custom DNA synthesis (GeneArt, Thermo Fisher Scientific) and ligated into XbaI/BamHI sites of 453

p20-Fc (CaMV35S promoter, used for co-localization analysis) (Suppl. Fig. S2) (Schoberer et al., 454

2009), pF (CaMV35S promoter, GALT fusions containing the catalytic domain of human 1,4-455

galactosyltransferase, used for the N-glycan processing assay) (Schoberer et al., 2014), p46 (UBQ10 456

promoter, expression of proteins containing a CTS region fused to the catalytic domain of NtGnTI and 457

GFP) (Schoberer et al., 2014), p49 (like p46 but with a C-terminal mRFP tag), p57 (386-bp weak 458

AtGnTI promoter, complementation of gntI plants with AtGnTI catalytic domain fused to GFP) 459

(Schoberer et al., 2014), p66 (UBQ10 promoter, expression of proteins containing a CTS region fused 460

to the catalytic domain of AtGnTI and GFP), p67 (1000-bp AtGnTI promoter, complementation of gntI 461

plants with AtGnTI catalytic domain fused to GFP), p68 (like p67 but with a C-terminal mRFP tag) 462

and p69 (like p66 but with a C-terminal mRFP tag). Plant expression vector p67 was generated by 463

insertion of the 1000-bp AtGnTI promoter region into the HindIII/XbaI site of p57. The 1000-bp 464

AtGnTI promoter region was amplified from Arabidopsis genomic DNA by PCR using 465

AthGnT33/AthGnTI_13R (Suppl. Table S2). p68 was generated by replacing GFP in vector p67 with 466

mRFP. p66 and p69 were generated by replacement of the AtGnTI promoter with the UBQ10 467

promoter. 468

The constructs for expression of the glycoprotein reporter (monoclonal antibody) (Strasser et al., 469

2009), ST-mRFP, ST-GFP, NtGnTI-CTS-GFP (Boevink et al., 1998; Schoberer et al., 2009), mRFP-470

AtCASP (Schoberer et al., 2010), NtGnTI-GFP, NtGnTI-RFP, ST-NtGnTI-GFP, ST-NtGnTI-mRFP 471

(Schoberer et al., 2014), aleu-GFP (Shin et al. 2017), mRFP-EMP12 (Shin et al., 2018), mRFP-SYP61 472

(Sanderfoot et al., 2001), GFP-RABF2b (Kotzer et al., 2004) and GFP-RABF1 (Ueda et al., 2001) 473

were all available from previous studies. p48-MUR3 was generated by PCR amplification from Col-0 474

cDNA of the MUR3 (AT2G20370) coding region using primers KAM1_1F/KAM1_3R. The PCR 475

product was XbaI/BglII digested and cloned into p48 (Hüttner et al., 2014). For GFP-ATG8e 476

expression, the coding region for Arabidopsis ATG8e (AT2G45170) was amplified by PCR from Col-477

0 cDNA using primers ATG8e_1F/ATG8e_2R. The PCR product was XbaI/BamHI digested and 478

cloned into XbaI/BamHI digested vector p37 (CaMV35S promoter). For the generation of p37, the 479



14

GFP coding region lacking the stop codon was PCR-amplified from the vector p20F using primers 480

GFP-14F/GFP-15R. The PCR product was SpeI/XbaI-digested and ligated into XbaI-linearized pPT2m 481

(Strasser et al., 2005) resulting in the N-terminal GFP expression vector p37. 482

NtGnTI and AtGnTI CTS regions with mutated single amino acids were generated by site-directed 483

mutagenesis using the Quikchange site directed mutagenesis protocol (Stratagene) and cloned into the 484

XbaI/BamHI sites of the used expression vectors. The expression construct p87 (MNS1 promoter, 485

MNS1 catalytic domain fused to GFP) for the chimeric MNS1 fusion proteins was described recently 486

(Veit et al., 2018). For expression of AtGnTI and AtGnTI-Q23A CTS regions, the corresponding 487

DNA was amplified with primers AthGnTI_14F/AthGnTI_16R and ligated into XbaI/BamHI digested 488

p87. 489

490

LC-ESI-MS analysis 491

Five-week-old N. benthamiana plants were used for Agrobacterium tumefaciens-mediated transient 492

expression of indicated constructs using the agroinfiltration technique as described previously 493

(Schoberer et al., 2009). The glycoprotein reporter was purified by affinity chromatography, subjected 494

to SDS-PAGE and Coomassie Brilliant Blue staining (Schoberer et al., 2014). The purified protein 495

band was excised from the gel, destained, carbamidomethylated, in-gel trypsin digested and analyzed 496

by liquid-chromatography electrospray ionization-mass spectrometry (LC-ESI-MS) (Stadlmann et al., 497

2008; Schoberer et al., 2009). A detailed explanation of N-glycan abbreviations and nomenclature can 498

be found at http://www.proglycan.com. 499

500

Complementation of Arabidopsis mutants 501

Arabidopsis gntI, cgl1 and mns1 mns2 mns3 mutants were transformed with different constructs by 502

floral dipping and selected on hygromycin (Strasser et al., 2004). The presence of the construct was 503

confirmed by PCR with specific primers. Proteins extracted from leaves of five-week-old plants were 504

subjected to SDS-PAGE under reducing conditions. Glycoproteins with processed N-glycans were 505

analyzed by immunoblotting with anti-horseradish peroxidase antibodies (anti-HRP, Sigma) that bind 506

to complex, truncated and hybrid N-glycans carrying 1,2-xylose and/or core 1,3-fucose residues 507

(Strasser et al., 2004; Jin et al., 2008; Kaulfürst-Soboll et al., 2011). MALDI-MS analysis was carried 508

out from 500 mg of leaves as described (Strasser et al., 2004). 509

510

Confocal imaging of fluorescent protein fusions 511

Leaves of five-week-old N. benthamiana plants were infiltrated with agrobacterium suspensions 512

carrying the protein(s) of interest with optical densities (OD600) from 0.05 to 0.2. Confocal images 513

were acquired 2–4 days post infiltration (dpi) on an upright Leica SP5 II confocal microscope using 514

the Leica LAS AF software system. GFP and mRFP were excited with the 488-nm and 561-nm laser 515

line, respectively, and detected at 500–530 nm and 600–630 nm, respectively. Dual-color image 516



15

acquisition of cells expressing both GFP and mRFP was performed simultaneously. Post-acquisition 517

image processing was performed in Adobe Photoshop CS5. 518

For co-localization analyses of co-expressed fluorescent protein fusions, images of latrunculin B-519

treated cells expressing the indicated NtGnTI and ST-CTS constructs together with the cis/medial-520

Golgi protein mRFP-AtCASP (Schoberer et al., 2010) or the trans-Golgi marker ST-mRFP (Boevink 521

et al., 1998) were acquired 2 dpi under non-saturating conditions using a 512 x 512 image format, 522

zoom factor 3 and a ×100/1.40 NA oil immersion objective of the Leica SP5. The pinhole was set to 1 523

airy unit, and background noise was reduced by 4–8 times line averaging. Only cells with comparable 524

GFP and mRFP fluorescence levels were considered for analysis. The images obtained were used for 525

co-localization analysis using the Pearson’s correlation coefficient. Calculations were made on 9–15 526

confocal images per co-expressed combination using the IMAGEJ 1.46 m plug-in JACOP (Bolte and 527

Cordelières, 2006). Statistical analyses were performed using a two-tailed Student’s t-test for the 528

comparison of two samples, assuming equal variances. FRET-FLIM data acquisition and analysis was 529

carried out as described previously (Schoberer et al., 2014). 530

531

Co-immunoprecipitation and immunoblotting 532

For analysis of AtGnTI-Q23A and AtGnTI interaction, AtGnTI-Q23A-mRFP (p69 vector) or AtGnTI-533

mRFP (p69 vector) was co-expressed with AtGnTI-GFP (p66 vector) by agroinfiltration of N. 534

benthamiana leaves. Two days after infiltration, the GFP-tagged protein was purified using GFP-Trap-535

A beads (ChromoTek) and co-purified protein was analyzed by immunoblotting with anti-mRFP 536

antibodies. NBR1 was detected with anti-NBR1 (Agrisera) and antibodies against actin (Agrisera) and 537

-tubulin (Sigma) were used as loading controls. 538

539

RT-qPCR analysis 540

RNA was extracted from 18-day-old Arabidopsis seedlings using the SV Total RNA Isolation System 541

(Promega) and the iScript cDNA Synthesis Kit (Bio-Rad, Vienna Austria). An aliquot of the cDNA 542

was used for the PCR amplification using iQ SYBR Green Supermix (Bio-Rad) with an iCycler (Bio-543

Rad) as described in detail previously (Hüttner et al., 2014). Expression of GFP-fusion proteins was 544

monitored using GFP-specific primers GFP-Q1F and GFP-Q2R and normalized to the UBQ5 545

transcript expression monitored with primers UBQ5_Q1F/UBQ5_Q2R. 546

547

Knockdown of N. benthamiana coatomer subunits 548

To knockdown the expression of the N. benthamiana COP and COP subunits, RNAi constructs were 549

generated as described (Shin et al., 2017). A synthetic DNA fragment consisting of intron 2 from 550

Arabidopsis 1,2-xylosyltransferase and an antisense DNA fragment corresponding to the coding 551

sequence for amino acids 35–186 of COP or 306–473 of COP were obtained by GeneArt gene 552

synthesis. The vector with the synthetic DNA was used as a template for PCR with primers Nb-eCOP-553



16

1F and Nb-eCOP-2R for COP and Nb-dCOP-1F and Nb-dCOP-2R for COP. The ‘sense’ PCR 554

product was digested with XbaI/KpnI and cloned into the synthetic DNA containing vector to generate 555

a sense–intron–antisense hairpin construct. The sense–intron–antisense sequence was subsequently 556

excised by XbaI/BamHI digestion and ligated into XbaI/BamHI digested plant expression vector 557

pPT2m. The pPT2-NbCOP-RNAi and pPT2-NbCOP-RNAi vectors were transformed into 558

agrobacteria and transiently expressed by agroinfiltration in N. benthamiana leaves together with the 559

fluorescent-tagged proteins. 560

561

Accession Numbers 562

Sequence data from this article can be found in the GenBank database under the following accession 563

numbers: M18769 (ST), AJ295993 (NtGnTI), in the Arabidopsis Information Resource under the 564

following accession numbers: AT4G38240 (AtGnTI), AT1G51590 (MNS1) and in the Nicotiana 565

benthamiana draft genome sequence v1.0.1 from the Sol Genomics Network: 566

Niben101Scf03249g01008 (COP) and Niben101Scf09552g01014 (COP). 567

568

Supplemental Data 569

Supplemental Figure S1. Amino acid sequence alignment of the N-terminal sequence (including the 570

TMD) from 74 putative plant GnTI sequences. 571

Supplemental Figure S2. CTS regions and used expression vectors. 572

Supplemental Figure S3. Subcellular localization of fusion proteins containing a chimeric or mutated 573

CTS region. 574

Supplemental Figure S4. Schematic illustration of the N-glycan processing assay. 575

Supplemental Figure S5. LC-ESI-MS analysis of the glycopeptide derived from a secreted 576

glycoreporter (IgG antibody) co-expressed with ST/GnTI-GALT, GnTI/ST-GALT or STFIYIQ-GALT. 577

Supplemental Figure S6. Complementation of the Arabidopsis gntI N-glycan processing defect. 578

Supplemental Figure S7. Complementation of Arabidopsis gntI and cgl1 plants. 579

Supplemental Figure S8. MALDI-TOF spectra of gntI expressing the indicated constructs under the 580

control of a weak AtGnTI promoter (386 bp). 581

Supplemental Figure S9. Co-localization of mutated AtGnTI with the trans-Golgi marker ST-582

GFPglyc. 583

Supplemental Figure S10. Comparison of the TMD sequence composition. 584

Supplemental Figure S11. AtGnTI variants with other conserved amino acids mutated reside in the 585

Golgi and are functional. 586

Supplemental Figure S12. Phenotypic comparison of complemented mns1 mns2 mns3 seedlings. 587

Supplemental Figure S13. Knockdown of the coatomer subunits COP or COP results in 588

mislocalization of Golgi-resident GnTI. 589

Supplemental Table S1. Statistical analyses of co-localization data. 590



17

Supplemental Table S2. List of used primers. 591

592

ACKNOWLEDGEMENTS 593

We thank Martina Dicker, Josephine Grass, Julia König, Daniel Maresch, Karin Polacsek and Yun-ji 594

Shin (all from BOKU) for technical assistance and help with N-glycan and glycopeptide analysis. 595

Verena Kriechbaumer (Oxford Brookes University) is acknowledged for supplying plants used at the 596

Central Laser Facility. This work was supported by grants from the Austrian Science Fund (FWF): 597

P23906-B20 (to R.S.), P28218-B22 (to R.S.), T655-B20 (to J.S.) and the Mizutani Foundation for 598

Glycosciences (to R.S.). We thank the STFC for funding access to the Central Laser Facility (grant no. 599

12130006 to Chris Hawes, Oxford Brookes University). We thank BOKU-VIBT Imaging Center for 600

access and expertise. 601

602

603

Table 1. FRET efficiency determined by FLIM 604

Donor Acceptor 𝝉D ± SD (ns) 𝝉DA ± SD (ns) Δ𝝉 (ns) E (%)

NtGnTI-CTS-GFP NtGnTI-CTS-mRFP 2.44 ± 0.06 2.08 ± 0.09 0.36 14.56

(n = 412) (n = 204)

NtGnTI-CTS-GFP ST-CTS-mRFP 2.44 ± 0.06 2.36 ± 0.06 0.08 3.35

(n = 412) (n = 238)

NtGnTI-CTS-GFP NtGnTI-CTS-Q25A-mRFP 2.44 ± 0.06 2.37 ± 0.06 0.07 2.89

(n = 412) (n = 273)

𝜏D, lifetime of the donor in the absence of the acceptor; 𝜏DA, lifetime of the donor in the presence of the 605 acceptor; Δ𝜏, lifetime contrast (𝜏D- 𝜏DA); E, FRET efficiency calculated according to (1-[𝜏D- 𝜏DA]) × 606 100; ns, nanosecond; SD, standard deviation; n, number of Golgi bodies analyzed. A minimum 607 decrease of the average excited-state fluorescence lifetime of the donor molecule by 0.20 ns or 8% in 608 the presence of the acceptor molecule was considered relevant to indicate interaction. 609 610

FIGURE LEGENDS 611

612

Figure 1. The exoplasmic half of the NtGnTI TMD contains a signal for cis/medial-Golgi 613

localization. (A) Sequence logo illustration of the degree of amino acid conservation within the TMD 614

of plant GnTI sequences (74 sequences in total). (B) Schematic illustration of the NtGnTI, ST, 615

ST/GnTI and GnTI/ST CTS regions. The amino acid sequence of the TMD is shown for each CTS 616

region. C: cytoplasmic tail, S: stem region. Sequences corresponding to the NtGnTI TMD are 617

highlighted in bold. (C) Co-localization analysis. NtGnTI-CTS-GFP, ST/GnTI-CTS-GFP and ST-618

CTS-GFP and GnTI/ST-GFP were co-expressed with mRFP-AtCASP (CASP) or ST-CTS-mRFP (ST) 619

(data are the mean ± SD, n > 10 confocal images). Asterisks indicate a significant difference between 620

CASP and ST localization (p ≤ 0.001, two tailed t-Test: two-sample assuming equal variances), ns: not 621

significant (p > 0.05). (D) Co-localization of NtGnTI-Q25A-GFP with mRFP-AtCASP or with ST-622

CTS-mRFP. Left images: scale bars = 5 µm, right images: scale bars = 1 µm. Magnified images of the 623



18

boxed region from the left images are shown in the right images. Graphs show relative fluorescence 624

intensities (NtGnTI-Q25A-GFP in green and mRFP-AtCASP in magenta) along the white lines (from 625

the right images). (E) Glycan processing assay. NtGnTI-Q25A-GALT (NtGnTI-Q25A) or the 626

indicated controls were co-expressed with a glycoprotein reporter. LC-ESI-MS analysis of the 627

glycopeptide “EEQYNSTYR” derived from the glycoprotein is shown. Reporter without GALT 628

(control), reporter co-expressed with NtGnTI-GALT (NtGnTI), ST-GALT (ST) or NtGnTI-Q25A-629

GALT. The schematic presentation of the major N-glycan structure is given. The symbols are drawn 630

according to the nomenclature from the Consortium for Functional Glycomics. For further details on 631

the abbreviation of the N-glycan structures see Supplemental Figure S4. 632

633

Figure 2. NtGnTI-Q25A locates in the Golgi, small puncta and the vacuole. Confocal microscopy 634

analysis was performed on N. benthamiana leaf epidermal cells expressing NtGnTI-Q25A fused to 635

mRFP or GFP and different subcellular markers. (A) Localization of NtGnTI-Q25A-mRFP was 636

monitored at different time points post-infiltration (2, 3 and 4 days post infiltration – dpi). (B) Co-637

localization with aleu-GFP (vacuolar marker). (C) Co-localization of NtGnTI-Q25A-GFP with 638

NtGnTI-mRFP reveals the labelling of small punctate structures (white arrows) that are distinct from 639

NtGnTI-labelled Golgi stacks. (D) Co-localization with mRFP-SYP61 (TGN-marker). (E) Co-640

localization with GFP-RABF2b (early endosome marker). (F) Co-localization with GFP-ATG8e 641

(autophagosome marker). (G) Co-localization with GFP-RABF1 (late endosome/multivesicular body 642

marker). Scale bars = 25 µm (A and B) and 10 µm (C to G). 643

644

Figure 3. AtGnTI-Q23A fusion protein expression does not restore the N-glycan processing 645

defect of gntI. (A) Immunoblot analysis of gntI AtGnTI:AtGnTI-GFP (WT) and gntI 646

AtGnTI:AtGnTI-Q23A-GFP (Q23A) seedlings with anti-HRP to detect plant complex N-glycans, with 647

GFP antibodies (anti-GFP) to detect the AtGnTI-GFP fusion protein and with antibodies against 648

ARGONAUTE 1 (anti-AGO) used as a loading control. (B) RT-pPCR analysis of transcripts from gntI 649

AtGnTI:AtGnTI-GFP (WT) and gntI AtGnTI:AtGnTI-Q23A-GFP (Q23A) seedlings. PCR for the 650

transgene expression was carried out with GFP-specific primers and normalized to UBQ5 transcript 651

expression. Data represent mean values ± SE, n = 3. (C) Immunoblot analysis of protein extracts from 652

gntI UBQ10:AtGnTI-mRFP (WT) or gntI UBQ10:AtGnTI-Q23A-mRFP (Q23A) with anti-HRP and 653

anti-RFP. (D) N-glycan analysis of complemented gntI expressing UBQ10:AtGnTI-mRFP (WT) or 654

UBQ10:AtGnTI-Q23A-mRFP (Q23A) by MALDI-MS. (E) Co-IP of AtGnTI (WT) and AtGnTI-655

Q23A (Q23A). UBQ10:AtGnTI-GFP co-expressed with UBQ10:AtGnTI-mRFP (WT) or 656

UBQ10:AtGnTI-Q23A-mRFP (Q23A) was purified with GFP-trap beads from N. benthamiana and 657

the co-purification of mRFP-fusion proteins was monitored by immunoblotting with mRFP antibodies. 658

(F) Localization of AtGnTI-mRFP, AtGnTI-Q23A-mRFP and ST-mRFP expressed in A. thaliana 659

seedlings under the control of the ubiquitin 10 promoter. Scale bars = 10 µm. 660



19

661

Figure 4. AtGnTI-Q23A-mRFP degradation is blocked in the presence of both MG132 and 662

concanamycin A. (A) CHX-degradation assay. 9-day-old Arabidopsis gntI UBQ10:AtGnTI-mRFP 663

(WT) and gntI UBQ10:AtGnTI-Q23A-mRFP (Q23A) seedlings were incubated for the indicated time 664

in 100 µg/ml cycloheximide (CHX) and subjected to SDS-PAGE and immunoblotting. The intact 665

fusion protein is marked by an arrowhead. Detection of actin was used as a control. (B) gntI 666

UBQ10:AtGnTI-mRFP (WT) and gntI UBQ10:AtGnTI-Q23A-mRFP (Q23A) seedlings were 667

incubated for 17 h with 20 µM MG132, (C) with 1 µM concanamycin A (ConcA) or (D) with 1 µM 668

ConcA and 20 µM MG132. AtGnTI expression was monitored with anti-RFP. NBR1 detection served 669

as a control for the inhibitor treatment (increased NBR1 signal) and tubulin as a loading control. The 670

quantitation shows the relative GnTI-mRFP amount normalized to tubulin expression. Values are 671

means ± SEM (n = 4 independent experiments). (E) gntI UBQ10:AtGnTI-mRFP (WT) and gntI 672

UBQ10:AtGnTI-Q23A-mRFP (Q23A) seedlings were incubated for 24 h with 20 µM of the class I -673

mannosidase inhibitor kifunensine (kif) and proteins were subjected to SDS-PAGE and 674

immunoblotting with anti-RFP antibodies. 675

676

Figure 5. AtGnTI variants with an altered amino acid at position 23 are mistargeted in plants 677

and do not complement the gntI N-glycan processing defect. (A) Confocal microscopy analysis in 678

N. benthamiana leaf epidermal cells of transiently expressed AtGnTI-Q23-mRFP mutants. Confocal 679

images were acquired 48 h after infiltration. (B) Transiently expressed AtGnTI-Q23-mRFP mutants 680

analyzed by immunoblotting using an anti-mRFP antibody. The quantitation shows the relative 681

amount of the upper (intact AtGnTI-mRFP) and the lower (AtGnTI-mRFP degradation product) 682

bands. Values are means ± SD (n = 4 independent experiments. The single amino acid labelling 683

indicates the substituted amino acid at position 23. (C) Immunoblot analysis of transgenic gntI plants 684

expressing the different AtGnTI-Q23-mRFP variants under the control of the endogenous AtGnTI 685

promoter. Protein extracts from leaves were analyzed with antibodies against complex plant N-glycans 686

(anti-HRP). Two independent lines are shown for each AtGnTI-Q23-mRFP variant, Col-0 (wt) and 687

gntI protein extracts were included as controls. Ponceau S staining of the membrane was used as a 688

loading control. 689

690

igure 6. Mistargeted MNS1-GFP does not complement the N-glycan processing defect and root 691

growth phenotype of mns1 mns2 mns3. MNS1:AtGnTI-MNS1-GFP and MNS1:AtGnTI-Q23A-692

MNS1-GFP were expressed in the Arabidopsis mns1 mns2 mns3 mutant. (A) Protein extracts were 693

analyzed from wild-type (Col-0), mns1 mns2 mns3 (123), mns1 mns2 mns3 complemented with 694

MNS1:AtGnTI-MNS1-GFP (WT) or MNS1:AtGnTI-Q23A-MNS1-GFP (Q23A) by SDS-PAGE and 695

immunoblotting using antibodies against complex plant N-glycans (anti-HRP) and GFP. Ponceau S 696

(Ponc.) staining of the membrane was used as a loading control. (B) Confocal microscopy of 697



20

Arabidopsis roots expressing the indicated constructs. Scale bars = 10 µm. (C) Phenotypic comparison 698

of 12-day-old Col-0, mns1 mns2 mns3 (123), mns1 mns2 mns3 complemented with MNS1:AtGnTI-699

MNS1-GFP (WT) or MNS1:AtGnTI-Q23A-MNS1-GFP (Q23A) seedlings grown on 0.5 x MS 700

supplemented with 1% sucrose. Scale bar = 1 cm. (D) Quantification of the root length from 8-day-old 701

seedlings grown on 0.5 x MS supplemented with 4% sucrose. For WT and Q23A the data from two 702

independent lines are shown. The mns3 single and the mns1 mns2 mns3 triple mutant are included as 703

controls. Data represent means ± SE (n > 50). 704

705

Figure 7. Knockdown of the coatomer subunits COP or COP results in mislocalization of 706

GnTI. (A) The left panel of images shows expression in the absence of any RNAi construct. “+COP” 707

indicates co-infiltration with the N. benthamiana COP RNAi construct. ”+COP” indicates co-708

infiltration with the N. benthamiana COP RNAi construct. mRFP-EMP12 (mRFP-tagged 709

Arabidopsis EMP12 protein lacking the N-terminal luminal domain), NtGnTI-mRFP, ST-NtGnTI-710

mRFP, AtGnTI-mRFP and AtGnTI-Q23A-mRFP were observed 48 h after infiltration using confocal 711

microscopy. Scale bars = 25 µm. (B) AtGnTI-Q23A-mRFP was treated with BFA (100 µg/ml) for the 712

indicated time. Scale bars = 25 µm. (C) Immunoblot analysis of NtGnTI-mRFP, ST-NtGnTI-mRFP, 713

AtGnTI-mRFP and AtGnTI-Q23A-mRFP. Proteins were co-expressed with the indicated COP RNAi 714

constructs, extracted 72 h after infiltration of N. benthamiana leaves and analyzed with anti-RFP 715

antibodies. An arrowhead marks the intact fusion protein. 716

717

Figure 8. Proposed working model for GnTI retrieval and its steady-state localization in the 718

cis/medial-Golgi. The model proposes a TMD-motif based sorting including the retrograde transport 719

from the trans-Golgi with COPI variants (e.g. COPIb) (Donohoe et al., 2007). The TMD sequence 720

motif with the conserved glutamine is either required for a specific protein-protein interaction with an 721

adaptor protein (indicated on the right side) or for interaction with a specific lipid class or Golgi 722

membrane environment (indicated on the left side) that partitions GnTI into retrograde transport 723

carriers to concentrate the glycosyltransferase in cis/medial-Golgi membranes. The large black arrows 724

indicate cargo that is transported from the ER trough the Golgi apparatus. 725

726

727



21

728

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Parsed CitationsAhn HK, Kang YW, Lim HM, Hwang I, Pai HS (2015) Physiological Functions of the COPI Complex in Higher Plants. Mol Cells 38: 866-875

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Aoki D, Lee N, Yamaguchi N, Dubois C, Fukuda MN (1992) Golgi retention of a trans-Golgi membrane protein, galactosyltransferase,requires cysteine and histidine residues within the membrane-anchoring domain. Proc Natl Acad Sci U S A 89: 4319-4323


Bakker H, Lommen A, Jordi W, Stiekema W, Bosch D (1999) An Arabidopsis thaliana cDNA complements the N-acetylglucosaminyltransferase I deficiency of CHO Lec1 cells. Biochem Biophys Res Commun 261: 829-832


Boevink P, Oparka K, Santa Cruz S, Martin B, Betteridge A, Hawes C (1998) Stacks on tracks: the plant Golgi apparatus traffics on anactin/ER network. Plant J 15: 441-447


Bolte S, Cordelières FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224: 213-232Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Burke J, Pettitt J, Humphris D, Gleeson P (1994) Medial-Golgi retention of N-acetylglucosaminyltransferase I. Contribution from alldomains of the enzyme. J Biol Chem 269: 12049-12059


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golgi localization of gnti requires a polar amino …...9 the golgi localization of gnti requires a...

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