Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2008 Jun 22.
Published in final edited form as: Mol Cell. 2007 Jun 22;26(6):821–830. doi: 10.1016/j.molcel.2007.05.015

Allele-Specific Suppression of a Defective Brassinosteroid Receptor Reveals an Essential Role of UDP-Glucose

Glycoprotein Glucosyltransferase for High-Fidelity ER Quality Control

Hua Jin 1, Zhenyan Yan 1, Kyoung Hee Nam 1,1, Jianming Li 1,2
PMCID: PMC1948852  NIHMSID: NIHMS26270  PMID: 17588517

SUMMARY

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a presumed folding sensor of protein quality control in the endoplasmic reticulum (ER). Previous biochemical studies with non-physiological substrates revealed that UGGT can glucosylate nonnative glycoproteins by recognizing subtle folding defects, however, its physiological function remains undefined. Here we show that mutations in the Arabidopsis EBS1 gene suppressed the growth defects of a brassinosteroid (BR) receptor mutant, bri1-9, in an allele-specific manner by restoring its BR sensitivity. Using a map-based cloning strategy, we discovered that EBS1 encodes the Arabidopsis homolog of UGGT. We demonstrated that bri1-9 is retained in the ER through interactions with several ER chaperones and that ebs1 mutations significantly reduce the stringency of the retention-based ER quality control, allowing export of the structurally-imperfect yet biochemically-competent bri1-9 to the cell surface for BR perception. Thus, our discovery provides the first genetic support for a physiological role of UGGT in high-fidelity ER quality control.

INTRODUCTION

Secretory and cell surface proteins play important roles in a variety of cell signaling processes throughout development of multicellular organisms. Because protein function strictly depends on its correct tertiary structure, eukaryotic cells are equipped with several ER quality control mechanisms to monitor protein folding, allowing export of only correctly folded proteins to their final destinations but retaining misfolded proteins in the ER (Ellgaard and Helenius, 2003). One of the best-studied ER quality control mechanisms is the calnexin (CNX)/calreticulin (CRT) cycle that relies on specific interaction between CNX/CRT, two ER-resident lectin-like chaperones, and Asn-linked monoglucosylated glycans (Glc1Man9GlcNAc2) (Helenius and Aebi, 2004; Moremen and Molinari, 2006). The CNX/CRT-Glc1Man9GlcNAc2 glycan interaction depends on the availability of the terminal glucose residue, which is initially generated through sequential removal of two glucose residues of the Glc3Man9GlcNAc2 core glycans on nascent proteins by glucosidases I and II. Eliminating the remaining glucose residue by glucosidase II releases the Man9GlcNAc2-containing glycoproteins from the ER lectins. A released glycoprotein that has successfully acquired its native conformation can exit the ER to continue its secretory journey. By contrast, a deglucosylated glycoprotein with an incompletely/improperly-folded conformation is recognized by a luminal enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) that transfers a glucose residue from UDP-glucose to Man9GlcNAc2 glycans. This UGGT-catalyzed reglucosylation promotes its reassociation with the ER lectins to initiate another round of CNX/CRT-mediated folding. The alternate action of glucosidase II and UGGT drives cycles of glycoprotein release from and binding to CNX/CRT until the glycoprotein is correctly folded. Terminally misfolded proteins are retrotranslocated into the cytosol for proteasome-mediated ER-associated degradation (McCracken and Brodsky, 1996).

UGGT is an ER luminal glycoprotein of ∼170 kDa consisting of two structurally independent domains: a variable N-terminal domain of ∼1,200 amino acids thought to participate in sensing protein folding and a highly-conserved C-terminal catalytic domain of ∼300 amino acids (Parodi, 2000). The presumed role of UGGT as a folding sensor in the CNX/CRT cycle was almost exclusively deduced from in vitro and cell culture studies using physiologically non-relevant substrates, which revealed that UGGT glucosylates only nonnative glycoproteins containing solvent-exposed hydrophobic residues (Trombetta, 2003). One notable exception is a cell-free microsomal study that implicated a role of the reglucosylation cycle in the folding of an endogenous protein, transferrin (Wada et al., 1997). Two recent in vitro biochemical studies have shown that UGGT was capable of discriminating minimally different conformations of its glycoprotein substrates, providing further support for a role of UGGT at the end of the folding process in retaining near-native folding intermediates (Caramelo et al., 2004; Taylor et al., 2004). However, up to now there is no genetic evidence for a physiological role of UGGT in CNX/CRT-mediated ER quality control despite availability of a yeast uggt mutant and a mouse embryonic fibroblast line lacking UGGT (Fanchiotti et al., 1998; Molinari et al., 2005).

BRI1 is a leucine-rich-repeat (LRR) receptor-like kinase (RLK) that functions as a cell surface receptor for brassinosteroids (BRs) (Kinoshita et al., 2005; Li and Chory, 1997), a unique class of plant polyhydroxylsteroids that are crucial for plant growth (Clouse and Sasse, 1998). Mutations in BRI1 or BR biosynthetic enzymes give rise to a set of characteristic mutant phenotypes that include dwarfism, reduced fertility, delayed flowering, altered vascular differentiation, and aberrant dark-grown morphology (Li and Chory, 1997; Li et al., 1996). Genetic and biochemical studies in recent years have revealed a linear signaling cascade that transduces an extracellular steroid signal into the nucleus (Li, 2005). BR binding to BRI1 promotes its dimerization with BAK1, a similar yet distinct LRR-RLK (Li et al., 2002; Nam and Li, 2002). Activation of both receptor kinases leads to inhibition of a GSK3-like kinase BIN2 (Li and Nam, 2002) and subsequent accumulation of non-phosphorylated BES1 and BZR1 inside the nucleus (Wang et al., 2002; Yin et al., 2002; Zhao et al., 2002) where they directly bind DNAs to regulate gene expression (He et al., 2005; Yin et al., 2005).

Because the extracellular domain of BRI1 is mainly composed of LRRs known to be involved in protein-protein interactions, it was previously thought that BRI1 requires a protein coligand to interact with BRs (Li and Chory, 1997). However, a recent study (Kinoshita et al., 2005) revealed that BRI1 can directly bind BRs via a 94-amino acid ligand-binding domain that is mutated in several known bri1 alleles, including bri1-6/bri1-119, bri1-7, bri1-113, and bri1-9 (Vert et al., 2005). It was predicted that these mutations destroy BRI1 receptor function by inhibiting BR binding (Friedrichsen et al., 2000; Li and Chory, 1997). However, it was shown recently that bri1-6 mutants contain a similar BR binding activity to wild-type plants, suggesting that other mechanisms are responsible for their mutant phenotypes (Kinoshita et al., 2005).

In this study, we made a surprising discovery that mutations in the Arabidopsis UGGT gene restore normal BR sensitivity to the bri1-9 mutant in an allele-specific manner. We demonstrate that the mutant phenotype of bri1-9 is caused by ER retention of a functionally competent BR receptor via the CNX/CRT-mediated ER quality control. Loss of the Arabidopsis UGGT activity prevents bri1-9 reglucosylation and CNX-bri1-9 interaction, thus significantly reducing the stringency of ER quality control. As a result, the structurally imperfect but biochemically active bri1-9 is exported to the cell surface where it can sense BRs and trigger a phosphorylation-mediated signaling cascade to promote plant growth. This unexpected finding provides the first genetic evidence for an in vivo role of UGGT in a high-fidelity ER quality control.

Results

The ebs1-1 Mutation Restores BR Sensitivity to the bri1-9 Mutant

To identify additional regulators of the BR signaling pathway, we conducted a genetic screen for extragenic suppressors of the bri1-9 mutation that changes Ser662 to Phe in the BR-binding domain and identified more than 80 wild-type looking seedlings (Figure 1A). One of them, named ebs1 for EMS-mutagenized bri1 suppressor 1, exhibits near wild-type morphology throughout its life cycle, including a large rosette, a long embryonic stem in the dark, and long floral stems at maturity (Figure 1B-1D). However, ebs1 fails to suppress the phenotypes of bin2 and det2 mutants (Figure 1E, 1F) defective in BR signaling and biosynthesis, respectively (Li et al., 1996; Li and Nam, 2002), indicating that ebs1 does not cause constitutive activation of BR signaling. We suspected that ebs1 might directly affect BR perception at the cell surface.

Figure 1.

Figure 1

Open in a new tab

ebs1 Suppresses bri1-9 Phenotype by Affecting BR Perception

(A) Schematic presentation of the genetic screen for ebs mutants in the bri1-9 background (see “Experimental Procedure” for details)

(B) Four-week-old soil-grown plants of bri1-9, wild type and ebs1-1 bri1-9.

(C) Seven-week-old mature plants of bri1-9, wild type and ebs1-1 bri1-9 grown in soil.

(D) The hypocotyl length of 4-day-old dark-grown seedlings of bri1-9, wild type and ebs1-1 bri1-9. Each bar represents the average measurement of ∼40 seedlings of two duplicate experiments. Error bar denotes standard error.

(E) bin2-1/+, bin2-1 and their corresponding double mutants with ebs1-1 grown in soil for 6 weeks.

(F) det2 and ebs1-1 det2 mutants grown in soil for 6 weeks.

Consistent with our prediction, the ebs1 mutation restored near-wild-type BR sensitivity to the bri1-9 mutant. As shown in Figure 2A, while bri1-9 roots are insensitive to brassinolide (BL), the most active BR, the elongation of ebs1 bri1-9 roots is inhibited by BL to a similar degree as wild type roots. We also examined BL-induced changes in the phosphorylation status of BES1, a robust biochemical indicator of BR signaling, using an anti-BES1 antibody (Mora-Garcia et al., 2004). As revealed in Figure 2B, upon BL treatment, BES1 was rapidly dephosphorylated in ebs1 bri1-9 mutants, resembling wild-type seedlings, whereas little conversion was observed in bri1-9 mutants. Restored BR sensitivity was also observed at the gene expression level. It is known that the Arabidopsis CPD gene is negatively regulated by BR (Mathur et al., 1998). Figure 2C shows that the CPD transcript is highly accumulated in bri1-9 and is slightly reduced by BL as compared to wild type. By contrast, the CPD transcript level is significantly reduced in ebs1-1 bri1-9 and gets even lower upon BL application.

Figure 2.

Figure 2

Open in a new tab

ebs1 Restores BR Sensitivity of bri1-9

(A) Quantitative analysis of BR sensitivity of bri1-9, wild type and ebs1-1 bri1-9 seedlings by the root inhibition assay as described previously (Li et al., 2001). Each data point represents the average root elongation of ∼40 seedlings of two duplicate experiments. Error bar denotes standard error.

(B) BR-induced changes in the phosphorylation status of BES1. 3-week-old seedlings of bri1-9, wild type, and ebs1-1 bri1-9 were treated with 1 μM BL for 1 hr in liquid half MS medium. Total proteins were extracted with 2X SDS buffer and separated by a 10% SDS-PAGE. BES1 was detected by anti-BES1 antibody (Mora-Garcia et al., 2004). Non-specific bands (*) were used as a loading control in the lower panel.

(C) Feedback inhibition of CPD expression by BL. Total mRNAs were extracted from BL-treated seedlings and probed for CPD as described previously (Li et al., 2001). The filter was stripped and reprobed with the 18S rDNA for loading control.

ebs1 Suppresses bri1-9 in an Allele-Specific Manner

Such a restored BR sensitivity could be caused by activation of an alternative BR receptor or by restoring the receptor function of bri1-9. To differentiate these two possibilities, we crossed the ebs1 mutation into several bri1 alleles of varying strength, including bri1-301, bri1-101, bri1-6, and bri1-113, the first two containing mutations in the kinase domain while the other two harboring mutations in the BR-binding domain (Vert et al., 2005). As shown in Figure 3, ebs1 fails to suppress any of the tested bri1 mutations, suggesting that ebs1 directly acts on bri1-9 to restore its receptor function.

Figure 3.

Figure 3

Open in a new tab

ebs1 is an Allele Specific Suppressor of the bri1-9 Mutation

(A) ebs1 cannot suppress bri1-301 and bri1-101 mutants containing a kinase domain mutation. From left to right: 6-week-old soil-grown plants of bri1-301, ebs1-1 bri1-301, bri1-101 and ebs1-3 bri1-101.

(B) ebs1 fails to suppress bri1-6 and bri1-113 mutants harboring mutations in the BR-binding domain. From left to right: 5-week-old soil-grown plants of bri1-6, ebs1-1 bri1-6, bri1-113, and ebs1-1 bri1-113.

EBS1 Encodes the Arabidopsis Homolog of UGGT

To understand the underlying biochemical mechanism by which ebs1 restores the BR receptor function of bri1-9, we isolated the EBS1 gene using a map-based cloning approach. We delimited the EBS1 locus to a 63-kb region at the bottom of chromosome 1 (Figure 4A). Sequence analysis of the entire 63-kb region of ebs1-1 identified a single nucleotide mutation within AT1g71220 changing the 35th exon/intron junction from AGgc to AGac that would cause a RNA splicing defect (Figure 4B). The identity of AT1g71220 as EBS1 was confirmed by our discovery that 4 other ebs1 alleles (ebs1-2 - ebs1-5) isolated from the same screen all contain a single-nucleotide change in AT1g71220 (Figure 4B). Further support came from our analysis of a double mutant carrying bri1-9 and a T-DNA insertional mutation in AT1g71220, which exhibits a similar morphology to ebs1-1 bri1-9 (Figure S1). RT-PCR and Western blot analyses confirmed splicing defects for ebs1-1 and ebs1-5 (Figure S2) and detected no EBS1 protein in the non-sense ebs1-2 and two splicing-defective mutants (Figure S3).

Figure 4.

Figure 4

Open in a new tab

Molecular Characterization of the EBS1 Gene

(A) Genetic mapping of EBS1. EBS1 was mapped to a 63-kb region at the bottom of chromosome I between markers CER469884 and F3I17_2, which is covered by two overlapping BAC clones: F23N20 and F3I17. Molecular makers and numbers of recombination for each marker are shown above and below the line, respectively. Sequence analysis of the entire 63-kb region of ebs1-1 identified a single nucleotide mutation in At1g71220 composed of 37 exons (bar) and 36 introns (line). Arrows indicate the positions of 5 ebs1 mutations.

(B) Summary of 5 ebs1 alleles, their predicted molecular defects and the corresponding positions in the human UGGT (accession # Q9NYU2).

AT1g71220, consisting of 37 exons and 36 introns, encodes a polypeptide of 1,613 amino acids with a 25-amino acid N-terminal signal peptide and a C-terminal ER retention signal KAEL, which is similar in sequence and domain organization to the ER luminal enzyme UGGT of yeast, Drosophila, and several vertebrates (Arnold et al., 2000; Fernandez et al., 1996; Parker et al., 1995; Tessier et al., 2000) (Figure S4). Like these UGGTs, the Arabidopsis enzyme contains a less conserved large N-terminal domain thought to be involved in folding sensing and a highly conserved catalytic C-terminal domain responsible for reglucosylation. It is interesting to note that the amino acids mutated in the two missense ebs1 alleles, ebs1-3 and ebs1-4, are absolutely conserved among the catalytic domains of all known UGGTs (Figure S4).

Database searches indicated that Arabidopsis contains only one UGGT enzyme. To test if ebs1 mutations affect plant growth, we crossed ebs1-1 into the BRI1+ background. We discovered that loss of UGGT has no obvious effect on plant growth (Figure S5), which is similar to the yeast Schizosaccharomyces pombe gpt1 mutant (Fanchiotti et al., 1998) but in sharp contrast to the uggt knockout mouse that exhibits an embryonic lethal phenotype (Molinari et al., 2005). The ebs1 mutation, however, does lead to unfolded protein response since the amounts of several ER proteins, including BiPs, PDIs, CNXs, and CRTs, are enhanced in ebs1-1 (Figure S5).

The bri1-9 Protein is Retained in the ER

Based on the molecular identity of the EBS1 gene, we hypothesized that the bri1-9 mutation causes a subtle conformational change so that it is recognized for reglucosylation by the Arabidopsis UGGT, resulting in its ER retention via Glc1Man9GlcNAc2-CNX/CRT interaction. We predicted that the ebs1 mutations disable this quality control mechanism, allowing bri1-9 move to the plasma membrane (PM) to perceive the BR signal.

To test our hypothesis, we examined the subcellular localization of bri1-9 by three different assays. First, we tested localization of bri1-9 by analyzing its sensitivity to endoglycosidase H (Endo H), which cleaves high mannose-type N-glycans of ER-localized proteins but fails to cleave Golgi-processed complex glycans. Total proteins of wild type or bri1-9 seedlings were treated with Endo H and analyzed by immunoblotting with an anti-BRI1 antibody (Mora-Garcia et al., 2004). As shown in Figure 5A, bri1-9 moves a bit slower than wild-type BRI1 of ∼150 kDa thought to contain 13 Asn-linked glycans. Such a mobility difference is caused by different glycoforms on the two BR receptors (Figure S6). Endo H treatment resulted in a slightly faster mobility of BRI1, most likely due to presence of one or more high mannose-type glycans on the mature BRI1, but generated a much smaller bri1-9 protein of ∼130 kDa consistent with complete deglycosylation. Next, we used the aqueous two-phase partitioning method (Larsson, 1983) to analyze the subcellular localization of bri1-9. Following partitioning of microsomal proteins, the relative distribution of BRI1 and bri1-9 into two different phases were analyzed by Western blotting using the anti-BRI1 antibody. As shown in Figure 5B and 5C, BRI1 was mainly observed in the upper phase that enriches two known PM markers, PMA2 (DeWitt et al., 1996) and PIP2 (Martre et al., 2002), while bri1-9 was detected only in the lower phase that enriches two known ER markers, BiP (Fontes et al., 1991) and PDI (Shorrosh et al., 1993). The ER localization of bri1-9 was further confirmed by our microscopic analysis of the bri1-9:GFP fusion protein. As shown in Figure 5E-5J, while the BRI1:GFP fusion protein is predominantly localized on the PM, bri1-9:GFP exhibits a localization pattern overlapping with that of a known ER-localized red fluorescent protein (DsRed:HDEL) tagged with the HDEL ER retention signal (Liu and Dixon, 2001). Taken together, our results clearly demonstrate that bri-9 is kept in the ER.

Figure 5.

Figure 5

Open in a new tab

bri1-9 is Retained in the ER but Moves to the PM in ebs1 Mutants

(A) Endo H sensitivity of BRI1 and bri1-9. Total proteins from bri1-9, wild type, ebs1-1 bri1-9 to ebs1-5 bri1-9 were subjected to Endo H treatment followed by immunoblotting with anti-BRI1 antiserum.

(B) to (D) Western blotting analysis of membrane fractions obtained through aqueous two phase partitioning of total microsomal proteins isolated from bri1-9 (B), wild type (C) and ebs1-3 bri1-9 (D) plants using antibodies against BRI1, BiP, PDI, PMA2, PIP2 and. M, U, and L indicate total microsomal fraction, upper phase that enriches PM proteins, and the lower phase that was depleted of PM, respectively.

(E) to (M) Confocal analysis of subcellular localization of BRI1:GFP and bri1-9:GFP in root tips of 6-day-old light-grown seedlings coexpressing DsRed:HDEL and BRI1:GFP (E-G) or bri1-9:GFP in either EBS1+ (H-J) or ebs1 (K-M) background.

The bri1-9 Protein Exits ER in the ebs1 Mutants

The three biochemical/microscopic assays were also used to examine whether bri1-9 is correctly localized to the PM in ebs1 mutants. As shown in Figure 5A, the Endo H assay indicates that ∼50% of the bri1-9 proteins becomes Endo-H resistant in the absence of UGGT activity, while the two-phase partitioning experiment (Figure 5D) demonstrates co-enrichment of a significant portion of bri1-9 with PM markers in the upper phase. Consistent with the two biochemical assays, examination of the green fluorescence pattern of bri1-9:GFP in the ebs1 background revealed a qualitative increase in the GFP signal on the PM (Figure 5K-5M). We thus conclude that ebs1 mutations significantly reduce the fidelity of retention-based ER quality control, allowing bri1-9 to be exported to the cell surface.

ebs1 Mutations Prevent Reglucosylation of bri1-9

To test if bri1-9 is monoglucosylated and if ebs1 prevents bri1-9 reglucosylation, we adopted the jack bean α-mannosidase (JBαM) assay that was previously used to demonstrate UGGT activity (Molinari et al., 2005; Ritter et al., 2005). This exoglycosidase removes terminal mannose residues from N-linked glycans (Figure 6A) and is thus capable of revealing the effect of ebs1 on the glucosylation status of ER-localized bri1-9. As shown in Figure 6B, JBαM treatment of the anti-GFP immunoprecipitate from BRI1:GFP transgenic plants slightly altered the mobility of the BRI1:GFP band, consistent with the presence of one or more high mannose-type glycans on mature BRI1, whereas a similar experiment with bri1-9:GFP EBS1+ transgenic plants resulted in a faster-moving bri1-9:GFP band. Consistent with the Endo H data, JBαM treatment of the anti-GFP immunoprecipitate of bri1-9:GFP ebs1-3 transgenic plants gave rise to two distinct bands: the top one with the same mobility as the JBαM-treated BRI1:GFP and the lower one moving even faster than the JBαM-treated bri1-9:GFP of the bri1-9:GFP EBS1+ transgenic plants. These results coupled with the data below strongly suggest that bri1-9 is monoglucosylated in the ER but becomes deglucosylated in ebs1 mutants.

Figure 6.

Figure 6

Open in a new tab

The ebs1 Mutation Inhibits Monoglucosylation of bri1-9

(A) Schematic illustration of the JBαM action. Hexagon, circle, and diamond represent glucose, mannose, and N-acetylglucosamine residues, respectively.

(B) Sensitivity of BRI1:GFP and bri1-9:GFP to JBαM treatment. Anti-GFP immunoprecipitates of BRI1:GFP, bri1-9:GFP, and bri1-9:GFP ebs1-3 transgenic plants were incubated with or without JBαM followed by Western blotting analysis with an anti-GFP antibody.

ebs1 Mutations Greatly Reduce the CNX-bri1-9 Interaction

It is known that a monoglucosylated glycoprotein is retained in the ER by interacting with CNX or CRT (Williams, 2006). Arabidopsis contains 2 CNX and 3 CRT homologs (Boyce et al., 1994; Huang et al., 1993; Persson et al., 2003). To examine whether bri1-9 interacts with any of these CNXs/CRTs, we performed a coimmunoprecipitation experiment with crude extracts of transgenic plants expressing BRI1:GFP or bri1-9:GFP using anti-GFP antibody. The presence of CNX or CRT in the resulting immunoprecipitates was revealed by Western blotting with an anti-maize CRT antibody (Pagny et al., 2000) that can detect Arabidopsis CNXs and CRTs (Persson et al., 2003). As shown in Figure 7, a strong band corresponding to CNXs was detected in the anti-GFP immunoprecipitate from transgenic plants expressing bri1-9:GFP but not from that of wild-type plants or the BRI1:GFP transgenic plants, providing additional support for monoglucosylation of bri1-9. The amount of co-immunoprecipitated CNX was reduced by ∼80% in the bri1-9:GFP ebs1 transgenic plants, indicating that the ebs1 mutation greatly reduced CNX-bri1-9 interaction. The residual amount of co-immunoprecipitated CNXs is most likely due to glycan-independent interaction between CNX and their clients (Williams, 2006).

Figure 7.

Figure 7

Open in a new tab

bri1-9 Interacts with CNXs and BiPs

Coimmunoprecipitation of bri1-9:GFP with BiPs and CNXs. Total protein crude extracts from wild type plants (lane 1), or transgenic plants of BRI1:GFP (lane 2), bri1-9:GFP (lane 3), or bri1-9:GFP ebs1-3 (lane 4) were immunoprecipitated with anti-GFP antibody and analyzed by Western blotting with antibodies against GFP, a maize CRT, or BiP. The left and right 4 lanes show the presence of BRI1:GFP or bri1-9:GFP, CNXs, CRTs, and BiPs in total protein extracts and anti-GFP immunoprecipitates, respectively. The numbers represent the relative signal intensity of co-immunoprecipitated CNXs or BiPs after normalization against the anti-GFP signal.

We also tested if BiP, another ER chaperone involved in ER retention of nonnative glycoproteins (Molinari et al., 2005), interacts with bri1-9. As shown in Figure 7, strong BiP signals were detected in the anti-GFP immunoprecipitates of both bri1-9:GFP and bri1-9:GFP ebs1-3 transgenic plants, explaining the ER retention of ∼50% of total bri1-9:GFP proteins in ebs1. Interestingly, the amount of coimmunoprecipitated BiP is slightly more in the ebs1-3 mutant background than in the wild-type background. This might be due to upregulation of BiP expression by the ebs1 mutation (Figure S5). Taken together, these results demonstrate that bri1-9 is retained in the ER via both Glc1Man9GlcNAc2-CNX and bri1-9-BiP interactions. The loss of UGGT activity prevents reglucosylation of bri1-9, resulting in escape of ∼50% of the conformationally-perturbed bri1-9 proteins from ER to the cell surface where they initiate the BR signaling cascade. We thus conclude that UGGT is crucial for a high-fidelity ER quality control in Arabidopsis that prevents export of misfolded glycoproteins.

DISCUSSION

UGGT is Essential for a High-Fidelity ER Quality Control

UGGT is generally believed to be the folding sensor in the CNX/CRT cycle because it is the only known component of the cycle capable of distinguishing between nonnative and native conformers of its substrates. Most of our knowledge on this unique selectivity of UGGT has been derived from in vitro biochemical studies using purified substrates, although a similar selectivity has also been observed in living cells (Trombetta, 2003). A recent study using mouse embryonic fibroblasts demonstrated that loss of the mouse UGGT activity prevented reglucosylation of a temperature-dependent folding mutant of the vesicular stomatitis virus G protein (tsO45 G) (Mollinari et al., 2005). Despite all these in vitro and cell culture studies, it remains unclear if UGGT has a physiological function in living cells or organisms. By screening extragenic suppressors of a BR receptor mutant, bri1-9, and cloning of the first suppressor gene, EBS1, we made a surprising discovery that reveals a physiological role of UGGT in retaining a misfolded glycoprotein in the ER and provides in vivo evidence that UGGT is capable of detecting minimally-altered structures.

We have shown here that UGGT is required for a stringent ER quality control in Arabidopsis. Mutations in the Arabidopsis UGGT gene allow the structurally-defective bri1-9 to escape from the ER and be correctly targeted to the cell-surface where it can function as a BR receptor, resulting in the allele-specific suppression of the bri1-9 mutation. Our conclusion contradicts that of the mouse study showing that deletion of the mouse UGT1 gene had no effect on the stringency of ER quality control (Molinari et al., 2005). The UGT1 deletion did lead to release of tsO45 G from the CNX/CRT cycle, but the misfolded viral protein was still trapped inside ER by a second level of retention-based quality control involving BiP-mediated formation of disulfide-bound protein aggregates. The apparent difference might be simply due to the nature of the folding-defective mutations of the two UGGT substrates. The bri1-9 mutation may cause only a minor structural defect that is sensitive to UGGT known to reglucosylate nearly-native folding intermediates (Caramelo et al., 2004), whereas the temperature-dependent folding-defect of tsO45 G can lead to a UGGT-non-recognizable conformation that spontaneously forms disulfide-linked protein aggregates. It is also possible that prolonged incubation at the nonpermissive temperature (39.5°C) and/or viral transfection activate the second level of retention-based quality control to prevent the export of misfolded viral protein.

The bri1-9 Phenotype is Caused by ER Retention of a Functional BR Receptor with a Subtle Structural Perturbation

Contrary to the previous prediction that bri1-9 affects ligand-binding or receptor dimerization (Friedrichsen et al., 2000), the results presented here revealed that the bri1-9 mutant phenotype is caused by an overzealous ER quality control that retains a functional BR receptor. The bri1-9 mutation changes Ser662 to Phe in the 22nd LRR that forms a minimal BR-binding motif with the so-called “70-amino-acid island” (Kinoshita et al., 2005). This Ser residue is highly conserved among the 25 LRRs of BRI1 and occupies the 8th position of the xL2xxL5xL7S8xN10x(L/F)12(S/T)13G14x(V/I)16P17xxΦ20xx(C/L)23x consensus motif (x represents any amino acid and Φ indicates hydrophobic residues) (Li and Chory, 1997). It is thought that most LRRs exhibit a curved horseshoe structure with a short parallel β-strand on the concave inner surface connected by a β-turn to an antiparallel 310-helical segment on the convex outside surface (Choe et al., 2005). The Ser residue, locating near the top of the concaved surface (L2xxL5xL7S8x), is predicted to be solvent-exposed and might participate in the formation of the hydrogen bond network that stabilizes the assembly of repeating LRR motifs (Choe et al., 2005). Replacement of this solvent-exposed Ser residue by a bulky hydrophobic Phe residue would cause a localized structural distortion that might perturb the packing of the 22nd LRR with the 70-amino-acid island, resulting in exposure of additional hydrophobic amino acids on the concaved inner surface, an important structural feature recognized by UGGT (Caramelo et al., 2003; Taylor et al., 2003). However, such a structural perturbation must be very subtle and cause no significant loss of the BRI1 function. The bri1-9 protein exhibited no detectable increase in protease sensitivity compared with BRI1 (data not shown) and the PM-localized bri1-9 is a functional BR receptor that can activate the BR signaling pathway. Thus, our results provide in vivo support for two recent in vitro experiments that demonstrated the capability of UGGT to glucosylate glycoproteins with near-native conformations (Caramelo et al., 2004; Taylor et al., 2004).

The Roles of CNXs/CRTs and BiPs in Retaining bri1-9

While UGGT puts a tag on a non-native glycoprotein, it is CNX or CRT that holds the tagged-misfolded protein in the ER (Williams, 2006). CNX is a type I ER membrane protein, whereas CRT is an ER luminal protein with an HDEL-type ER retrieval signal. Both proteins share a similar domain organization and 3-D structure, exhibit identical lectin binding activity, and have overlapping yet distinct client glycoproteins (Williams, 2006). The Arabidopsis genome encodes two CNX (Boyce et al., 1994; Huang et al., 1993) and three CRT homologs (Persson et al., 2003). Our results show that bri1-9 interacts with CNXs only and that the CNX-bri1-9 interaction is largely dependent on the presence of monoglucosylated glycans since the interaction was greatly reduced in the ebs1 mutant. Nevertheless, a residual amount of CNXs was found to be coimmunoprecipitated with bri1-9 in the absence of UGGT (Figure 7). This interesting result provides support for the idea that CNX is a bona fide molecular chaperone interacting directly with the protein moiety of nonnative glycoproteins (Williams, 2006).

The fact that ∼50% of the bri1-9 proteins are still retained in the ER in ebs1 mutants strongly suggests that other ER proteins might also be involved in trapping bri1-9 in the ER. Our coimmunoprecipitation experiment suggests that BiPs might be responsible for retaining the other half of the bri1-9 proteins in the folding compartment (Figure 7). Arabidopsis contains 3 BiPs that are upregulated by the “unfolded protein response” pathway to deal with various ER stresses (Martinez and Chrispeels, 2003; Noh et al., 2003). BiP is known to interact with misfolded or incompletely-assembled proteins by recognizing hydrophobic patches that are normally buried inside native proteins, thus facilitating protein folding and maintaining the solubility of terminally-misfolded proteins (Fewell et al., 2001). A recent study suggested that BiP is involved in the second phase of retention-based ER quality control when terminally-misfolded proteins form disulfide-bound protein aggregates (Molinari et al., 2005). This raises the question whether the BiP-retained bri1-9 forms similar protein aggregates in bri1-9 or ebs1 bri1-9 mutants. However, separation of immunoprecipitated bri1-9 by non-reducing SDS-PAGE failed to reveal the presence of disulfide-linked bri1-9 aggregates (data not shown). It remains to be determined if BiP-retained bri1-9 is targeted for ER-associated degradation or undergoes additional rounds of facilitated protein folding.

In conclusion, our results demonstrate that UGGT is a crucial component of a high-fidelity ER quality control in Arabidopsis that prevents the export of a structurally perturbed but functionally active BR receptor. The retention of biochemically-active glycoproteins by an overzealous ER quality control is known to be responsible for a severe clinical phenotype of the most common cystic fibrosis mutation, ΔF508 (occurring in ∼80% cystic fibrosis patients), which causes the CNX-mediated ER retention of a partially active cAMP-dependent chloride channel missing the amino acid Phe508, known as cystic fibrosis transmembrane conductance regulator (CFTR) (Sanders and Myers, 2004). Overcoming such a retention-based ER quality control mechanism, such as reducing the ER Ca2+ level to inhibit CFTR-CNX interaction or using chemical chaperones to facilitate ΔF508-CFTR folding, can lead to the correct targeting of the mutated yet functional CFTR to the apical membrane of the epithelial cells (Brown et al., 1996; Egan et al., 2002), thus providing a promising clinical cure for the fatal genetic disease. Our discovery that bri1-9 is a physiological substrate for UGGT coupled with the isolation of more than 80 ebs mutants makes the Arabidopsis dwarf mutant an excellent genetic model system to investigate ER quality control in a multicellular eukaryotic organism. Molecular cloning of additional EBS genes and biochemical characterization of their gene products will increase our understanding of the molecular mechanism of retention-based ER quality control, which could lead to better therapeutic strategies for treating cystic fibrosis and other protein folding diseases.

EXPERIMENTAL PROCEDURES

Plant Materials and Growth Conditions

Arabidopsis thaliana ecotype Columbia (Col-0) was the parental strain for all mutants and transgenic lines with the exception of bri1-6 [Enkheim (En2)] and bri1-9 [Wassilewskija (WS)] used for genetic mapping. Methods for seed sterilization and conditions for plant growth were described previously (Li et al., 2001).

Isolation of bri1-9 (Col) and bri1-9 Suppressor Mutants

The bri1-9 (Col) mutant was isolated from the genetic screen that identified the bin2-2 mutant (Li and Nam, 2002). bri1-9 (Col) seeds were mutagenized with 0.4% ethyl methanesulfonate (Sigma-Aldrich) as described (Yin et al., 2002). Approximately 150,000 M2 seeds derived from 15,000 M1 plants were screened on synthetic medium (1/2 MS salt plus 1% sucrose). After 4-week growth under a 16 h-light/8 h-dark growth condition, wild-type looking seedlings were picked and transferred into soil for continued growth and seed collection. The candidate suppressors were genotyped using a bri1-9 dCAPS marker (see TableS1 for details) to eliminate pollen or seed contamination.

Cloning of the EBS1 Gene

The ebs1-1 bri1-9 (Col) mutant was crossed with bri1-9 (Ws) and the F1 plants were self-fertilized. The resulting F2 plants were grown on synthetic medium for 4 weeks and the seedlings exhibiting the suppressed-bri1-9 phenotype were analyzed with molecular markers (see Table S1 for details). A total of 3,400 chromosomes were scored to narrow the EBS1 locus to a 63-kb region covered by two overlapping BAC clones (F23N20 and F3I17) on the bottom of Chromosome I. The entire 63-kb region of ebs1-1 was sequenced and the resulting sequences were compared with the published sequences to identify a single nucleotide change. The bri1-9 suppressors showing similar morphology to ebs1-1 bri1-9 were individually crossed with bri1-9 (Ws) and the resulting F1 hybrids were self-fertilized. Thirty to fifty plants exhibiting the suppressed-bri1-9 phenotype of each F2 population were selected for mapping analysis. Mutants exhibiting a tight linkage between their suppressor loci with EBS1 were chosen to sequence the entire EBS1 gene.

Generation of Transgenic Plants

The construct, pPZP212-BRI1-BRI1:GFP, used for generating BRI1:GFP transgenic plants was described previously (Friedrichsen et al., 2000). The bri1-9 mutation was introduced into the pPZP212-BRI1-BRI1:GFP transgene by site-directed mutagenesis using the Stratagene’s QuickChange II XL site-directed mutagenesis kit to generate the pPZP212-BRI1-bri1-9:GFP construct. Both constructs were individually transformed into wild-type plants using the Agrobacterium-mediated method. The pPZP212-BRI1-bri1-9:GFP ebs1 plant was generated by crossing a selected pPZP212-BRI1-bri1-9:GFP transgenic line with ebs1-3. The 35S-DsRed:HDEL construct was created by cloning the entire coding region of DsRed (Clontech) into binary vector pCHF1 (Fankhauser et al., 1999) and adding the HDEL-ER retrieval signal at the C-terminus of DsRed through sire-directed mutagenesis, and transformed into appropriate transgenic lines expressing BRI1GFP or bri1-9GFP.

Two Phase Partitioning

Microsomal membranes were isolated from 4-week-old Arabidopsis plants according to a previously described protocol (Hong et al., 1999) and further separated into PM and intracellular membrane fractions by the aqueous two-phase partitioning method (Larsson, 1983). Equal amounts of proteins of the total microsomal fraction, the PM-enriched upper phase, and the PM-depleted lower phase were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblotting with anti-BRI1 (Mora-Garcia et al., 2004), anti-PMA2 (Morsomme et al., 1998), anti-PIP2 (Martre et al., 2002), anti-BiP (SPA-818, Stressgen, BC, Canada), and anti-PDI (Rose Biotechnology Inc. Winchendon, MA) antibodies.

Co-immunoprecipitation

Total protein crude extracts were prepared from 3-week-old seedlings grown on synthetic medium. Seedlings were ground in liquid nitrogen and extracted with 5 mL/g tissue of the extraction buffer (50 mM Tris pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.2% Triton X-100, 10% glycerol and the protease inhibitor cocktail containing 1 mM phenylmethylsulphonyl fluoride plus 2 μg/mL each of aprotinin, leupeptin, and pepstatin A [all purchased from Fisher Scientific]). The lysates were cleared by centrifugation at 5,000g for 5 min at 4°C. The supernatant was incubated with a polyclonal anti-GFP antibody (TP401, Torrey Pines Biolabs, Texas) for 1 hr at 4°C and immunoprecipitated with Protein A-agarose beads (Invitrogen) for 1 hr at 4°C. The resulting immunoprecipitates were washed three times with the extraction buffer, separated on a 7.5% SDS-PAGE gel, and analyzed by Western blotting using anti-GFP, anti-BiP, or anti-maize-CRT antibody (Pagny et al., 2000). For the anti-CRT antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG light-chain (Jackson Immunology) was used as the secondary antibody to avoid interference from IgG heavy chain.

Deglycosylation Assays

The Endo H assay was performed according to the manufacturer’s recommended procedure using protein crude extracts of 4-week-old seedlings. For the JBαM assay, anti-GFP immunoprecipitates of BRI1:GFP or bri1-9:GFP-expressing transgenic lines were denatured in 1% SDS, diluted to 0.2% SDS with 50 mM sodium citrate buffer (pH 4.5) containing 1% Triton X-100, and incubated overnight at 37°C with or without 330 mU JBαM (MP Biomedicals). Treated proteins were separated by SDS-PAGE and analyzed by Western blotting with anti-BRI1 (Endo H assay) or anti-GFP (JBαM assay) antiserum.

Confocal Microscopy

The localization patterns of BRI1:GFP, bri1-9:GFP, and DsRed:HDEL were examined by imaging root tips of 6-day-old seedlings of BRI1:GFP DsRed:HDEL, bri1-9:GFP DsRed:HDEL, and bri1-9:GFP DsRed:HDEL ebs1 double transgenic lines with a Zeiss LSM510 confocal microscope using 100X water immersion objective. GFP signal (excitation at 488 nm with emission at 505-530 nm) and DsRed (excitation at 543 nm and emission at >585 nm) were acquired sequentially.

Supplementary Material

01

ACKNOWLEDGEMENT

We are grateful to Drs. R. Boston, M. Boutry, M. Chrispeel, J. Chory, Y. Yin, F. Tax, and Z. Wang, and the Arabidopsis Biological Resource Center (Ohio State University, Columbus) for antibodies and seeds. We thank Drs. Kenneth Cadigan, Amy Chang, Steven Clark, Randal Kaufman, and Yanzhuang Wang, and members of the Li lab for helpful discussions and critical comments on the manuscript. This work was supported in part by a National Institutes of Health grant (GM060519) and a Department of Energy grant (ER15673).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  1. Arnold SM, Fessler LI, Fessler JH, Kaufman RJ. Two homologues encoding human UDP-glucose:glycoprotein glucosyltransferase differ in mRNA expression and enzymatic activity. Biochemistry. 2000;39:2149–2163. doi: 10.1021/bi9916473. [DOI] [PubMed] [Google Scholar]
  2. Boyce JM, Coates D, Fricker MD, Evans DE. Genomic sequence of a calnexin homolog from Arabidopsis thaliana. Plant Physiol. 1994;106:1691. doi: 10.1104/pp.106.4.1691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown CR, Hong-Brown LQ, Biwersi J, Verkman AS, Welch WJ. Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones. 1996;1:117–125. doi: 10.1379/1466-1268(1996)001<0117:ccctmp>2.3.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caramelo JJ, Castro OA, Alonso LG, De Prat-Gay G, Parodi AJ. UDP-Glc:glycoprotein glucosyltransferase recognizes structured and solvent accessible hydrophobic patches in molten globule-like folding intermediates. Proc Natl Acad Sci U S A. 2003;100:86–91. doi: 10.1073/pnas.262661199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Caramelo JJ, Castro OA, de Prat-Gay G, Parodi AJ. The endoplasmic reticulum glucosyltransferase recognizes nearly native glycoprotein folding intermediates. J Biol Chem. 2004;279:46280–46285. doi: 10.1074/jbc.M408404200. [DOI] [PubMed] [Google Scholar]
  6. Choe J, Kelker MS, Wilson IA. Crystal structure of human toll-like receptor 3 (TLR3) ectodomain. Science. 2005;309:581–585. doi: 10.1126/science.1115253. [DOI] [PubMed] [Google Scholar]
  7. Clouse SD, Sasse JM. BRASSINOSTEROIDS: Essential Regulators of Plant Growth and Development. Annu Rev Plant Physiol Plant Mol Biol. 1998;49:427–451. doi: 10.1146/annurev.arplant.49.1.427. [DOI] [PubMed] [Google Scholar]
  8. DeWitt ND, Hong B, Sussman MR, Harper JF. Targeting of two Arabidopsis H(+)-ATPase isoforms to the plasma membrane. Plant Physiol. 1996;112:833–844. doi: 10.1104/pp.112.2.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Egan ME, Glockner-Pagel J, Ambrose C, Cahill PA, Pappoe L, Balamuth N, Cho E, Canny S, Wagner CA, Geibel J, Caplan MJ. Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells. Nat Med. 2002;8:485–492. doi: 10.1038/nm0502-485. [DOI] [PubMed] [Google Scholar]
  10. Ellgaard L, Helenius A. Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol. 2003;4:181–191. doi: 10.1038/nrm1052. [DOI] [PubMed] [Google Scholar]
  11. Fanchiotti S, Fernandez F, D′Alessio C, Parodi AJ. The UDP-Glc:Glycoprotein glucosyltransferase is essential for Schizosaccharomyces pombe viability under conditions of extreme endoplasmic reticulum stress. J Cell Biol. 1998;143:625–635. doi: 10.1083/jcb.143.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fankhauser C, Yeh KC, Lagarias JC, Zhang H, Elich TD, Chory J. PKS1, a substrate phosphorylated by phytochrome that modulates light signaling in Arabidopsis. Science. 1999;284:1539–1541. doi: 10.1126/science.284.5419.1539. [DOI] [PubMed] [Google Scholar]
  13. Fernandez F, Jannatipour M, Hellman U, Rokeach LA, Parodi AJ. A new stress protein: synthesis of Schizosaccharomyces pombe UDP--Glc:glycoprotein glucosyltransferase mRNA is induced by stress conditions but the enzyme is not essential for cell viability. Embo J. 1996;15:705–713. [PMC free article] [PubMed] [Google Scholar]
  14. Fewell SW, Travers KJ, Weissman JS, Brodsky JL. The action of molecular chaperones in the early secretory pathway. Annu Rev Genet. 2001;35:149–191. doi: 10.1146/annurev.genet.35.102401.090313. [DOI] [PubMed] [Google Scholar]
  15. Fontes EB, Shank BB, Wrobel RL, Moose SP, GR OB, Wurtzel ET, Boston RS. Characterization of an immunoglobulin binding protein homolog in the maize floury-2 endosperm mutant. Plant Cell. 1991;3:483–496. doi: 10.1105/tpc.3.5.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Friedrichsen DM, Joazeiro CA, Li J, Hunter T, Chory J. Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase. Plant Physiol. 2000;123:1247–1256. doi: 10.1104/pp.123.4.1247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. He JX, Gendron JM, Sun Y, Gampala SS, Gendron N, Sun CQ, Wang ZY. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science. 2005;307:1634–1638. doi: 10.1126/science.1107580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Helenius A, Aebi M. Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem. 2004;73:1019–1049. doi: 10.1146/annurev.biochem.73.011303.073752. [DOI] [PubMed] [Google Scholar]
  19. Hong B, Ichida A, Wang Y, Gens JS, Pickard BG, Harper JF. Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum. Plant Physiol. 1999;119:1165–1176. doi: 10.1104/pp.119.4.1165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huang L, Franklin AE, Hoffman NE. Primary structure and characterization of an Arabidopsis thaliana calnexin-like protein. J Biol Chem. 1993;268:6560–6566. [PubMed] [Google Scholar]
  21. Kinoshita T, Cano-Delgado A, Seto H, Hiranuma S, Fujioka S, Yoshida S, Chory J. Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1. Nature. 2005;433:167–171. doi: 10.1038/nature03227. [DOI] [PubMed] [Google Scholar]
  22. Larsson C. Partition in aqueous polymer two-phase systems: a rapid method for separation of membrane particles according to their surface properties. In: Hall JM, Moore AL, editors. Isolation of Membranes and Organelles from Plant Cells. Academic Press; London: 1983. pp. 277–309. [Google Scholar]
  23. Li J. Brassinosteroid signaling: from receptor kinases to transcription factors. Curr Opin Plant Biol. 2005;8:526–531. doi: 10.1016/j.pbi.2005.07.009. [DOI] [PubMed] [Google Scholar]
  24. Li J, Chory J. A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell. 1997;90:929–938. doi: 10.1016/s0092-8674(00)80357-8. [DOI] [PubMed] [Google Scholar]
  25. Li J, Nagpal P, Vitart V, McMorris TC, Chory J. A role for brassinosteroids in light-dependent development of Arabidopsis. Science. 1996;272:398–401. doi: 10.1126/science.272.5260.398. [DOI] [PubMed] [Google Scholar]
  26. Li J, Nam KH. Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase. Science. 2002;295:1299–1301. doi: 10.1126/science.1065769. [DOI] [PubMed] [Google Scholar]
  27. Li J, Nam KH, Vafeados D, Chory J. BIN2, a new brassinosteroid-insensitive locus in Arabidopsis. Plant Physiol. 2001;127:14–22. doi: 10.1104/pp.127.1.14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell. 2002;110:213–222. doi: 10.1016/s0092-8674(02)00812-7. [DOI] [PubMed] [Google Scholar]
  29. Liu CJ, Dixon RA. Elicitor-induced association of isoflavone O-methyltransferase with endomembranes prevents the formation and 7-O-methylation of daidzein during isoflavonoid phytoalexin biosynthesis. Plant Cell. 2001;13:2643–2658. doi: 10.1105/tpc.010382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Martinez IM, Chrispeels MJ. Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes. Plant Cell. 2003;15:561–576. doi: 10.1105/tpc.007609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Martre P, Morillon R, Barrieu F, North GB, Nobel PS, Chrispeels MJ. Plasma membrane aquaporins play a significant role during recovery from water deficit. Plant Physiol. 2002;130:2101–2110. doi: 10.1104/pp.009019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mathur J, Molnar G, Fujioka S, Takatsuto S, Sakurai A, Yokota T, Adam G, Voigt B, Nagy F, Maas C. Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids. Plant J. 1998;14:593–602. doi: 10.1046/j.1365-313x.1998.00158.x. [DOI] [PubMed] [Google Scholar]
  33. McCracken AA, Brodsky JL. Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP. J Cell Biol. 1996;132:291–298. doi: 10.1083/jcb.132.3.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Molinari M, Galli C, Vanoni O, Arnold SM, Kaufman RJ. Persistent glycoprotein misfolding activates the glucosidase II/UGT1-driven calnexin cycle to delay aggregation and loss of folding competence. Mol Cell. 2005;20:503–512. doi: 10.1016/j.molcel.2005.09.027. [DOI] [PubMed] [Google Scholar]
  35. Mora-Garcia S, Vert G, Yin Y, Cano-Delgado A, Cheong H, Chory J. Nuclear protein phosphatases with Kelch-repeat domains modulate the response to brassinosteroids in Arabidopsis. Genes Dev. 2004;18:448–460. doi: 10.1101/gad.1174204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moremen KW, Molinari M. N-linked glycan recognition and processing: the molecular basis of endoplasmic reticulum quality control. Curr Opin Struct Biol. 2006;16:592–599. doi: 10.1016/j.sbi.2006.08.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Morsomme P, Dambly S, Maudoux O, Boutry M. Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region. J Biol Chem. 1998;273:34837–34842. doi: 10.1074/jbc.273.52.34837. [DOI] [PubMed] [Google Scholar]
  38. Nam KH, Li J. BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell. 2002;110:203–212. doi: 10.1016/s0092-8674(02)00814-0. [DOI] [PubMed] [Google Scholar]
  39. Noh SJ, Kwon CS, Oh DH, Moon JS, Chung WI. Expression of an evolutionarily distinct novel BiP gene during the unfolded protein response in Arabidopsis thaliana. Gene. 2003;311:81–91. doi: 10.1016/s0378-1119(03)00559-6. [DOI] [PubMed] [Google Scholar]
  40. Pagny S, Cabanes-Macheteau M, Gillikin JW, Leborgne-Castel N, Lerouge P, Boston RS, Faye L, Gomord V. Protein recycling from the Golgi apparatus to the endoplasmic reticulum in plants and its minor contribution to calreticulin retention. Plant Cell. 2000;12:739–756. doi: 10.1105/tpc.12.5.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Parker CG, Fessler LI, Nelson RE, Fessler JH. Drosophila UDP-glucose:glycoprotein glucosyltransferase: sequence and characterization of an enzyme that distinguishes between denatured and native proteins. Embo J. 1995;14:1294–1303. doi: 10.1002/j.1460-2075.1995.tb07115.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Parodi AJ. Protein glucosylation and its role in protein folding. Annu Rev Biochem. 2000;69:69–93. doi: 10.1146/annurev.biochem.69.1.69. [DOI] [PubMed] [Google Scholar]
  43. Persson S, Rosenquist M, Svensson K, Galvao R, Boss WF, Sommarin M. Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants. Plant Physiol. 2003;133:1385–1396. doi: 10.1104/pp.103.024943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ritter C, Quirin K, Kowarik M, Helenius A. Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT. Embo J. 2005;24:1730–1738. doi: 10.1038/sj.emboj.7600645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sanders CR, Myers JK. Disease-related misassembly of membrane proteins. Annu Rev Biophys Biomol Struct. 2004;33:25–51. doi: 10.1146/annurev.biophys.33.110502.140348. [DOI] [PubMed] [Google Scholar]
  46. Shorrosh BS, Subramaniam J, Schubert KR, Dixon RA. Expression and Localization of Plant Protein Disulfide Isomerase. Plant Physiol. 1993;103:719–726. doi: 10.1104/pp.103.3.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Taylor SC, Ferguson AD, Bergeron JJ, Thomas DY. The ER protein folding sensor UDP-glucose glycoprotein-glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation. Nat Struct Mol Biol. 2004;11:128–134. doi: 10.1038/nsmb715. [DOI] [PubMed] [Google Scholar]
  48. Taylor SC, Thibault P, Tessier DC, Bergeron JJ, Thomas DY. Glycopeptide specificity of the secretory protein folding sensor UDP-glucose glycoprotein:glucosyltransferase. EMBO Rep. 2003;4:405–411. doi: 10.1038/sj.embor.embor797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tessier DC, Dignard D, Zapun A, Radominska-Pandya A, Parodi AJ, Bergeron JJ, Thomas DY. Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme. Glycobiology. 2000;10:403–412. doi: 10.1093/glycob/10.4.403. [DOI] [PubMed] [Google Scholar]
  50. Trombetta ES. The contribution of N-glycans and their processing in the endoplasmic reticulum to glycoprotein biosynthesis. Glycobiology. 2003;13:77R–91R. doi: 10.1093/glycob/cwg075. [DOI] [PubMed] [Google Scholar]
  51. Vert G, Nemhauser JL, Geldner N, Hong F, Chory J. Molecular mechanisms of steroid hormone signaling in plants. Annu Rev Cell Dev Biol. 2005;21:177–201. doi: 10.1146/annurev.cellbio.21.090704.151241. [DOI] [PubMed] [Google Scholar]
  52. Wada I, Kai M, Imai S, Sakane F, Kanoh H. Promotion of transferrin folding by cyclic interactions with calnexin and calreticulin. Embo J. 1997;16:5420–5432. doi: 10.1093/emboj/16.17.5420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wang ZY, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y, Fujioka S, Yoshida S, Asami T, Chory J. Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell. 2002;2:505–513. doi: 10.1016/s1534-5807(02)00153-3. [DOI] [PubMed] [Google Scholar]
  54. Williams DB. Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum. J Cell Sci. 2006;119:615–623. doi: 10.1242/jcs.02856. [DOI] [PubMed] [Google Scholar]
  55. Yin Y, Vafeados D, Tao Y, Yoshida S, Asami T, Chory J. A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis. Cell. 2005;120:249–259. doi: 10.1016/j.cell.2004.11.044. [DOI] [PubMed] [Google Scholar]
  56. Yin Y, Wang ZY, Mora-Garcia S, Li J, Yoshida S, Asami T, Chory J. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell. 2002;109:181–191. doi: 10.1016/s0092-8674(02)00721-3. [DOI] [PubMed] [Google Scholar]
  57. Zhao J, Peng P, Schmitz RJ, Decker AD, Tax FE, Li J. Two putative BIN2 substrates are nuclear components of brassinosteroid signaling. Plant Physiol. 2002;130:1221–1229. doi: 10.1104/pp.102.010918. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS26270-supplement-01.pdf (860.9KB, pdf)

RESOURCES