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N-glycosylation has a great impact on glycoprotein structure, conformation, stability, solubility, immunogenicity and enzyme activity. Structural characterization of N-glycoproteome has been challenging but can provide insights into the extent of protein folding and surface topology. We describe a highly sensitive proteomics method for large-scale identification and quantification of glycoproteins in Arabidopsis through (15) N-metabolic labeling, selective enrichment of glycopeptides, data-dependent MS/MS analysis and automated database searching. In-house databases of Arabidopsis glycoproteins and glycopeptides containing Asn-X-Ser/Thr/Cys motifs were constructed by reducing 20% and 90% of the public database size, respectively, to enable a rapid analysis of large datasets for comprehensive identification and quantification of glycoproteins and heterogeneous N-glycans in a complex mixture. Proteome-wide analysis identified c. 100 stress-related N-glycoproteins, of which the endoplasmic reticulum (ER) resident proteins were examined to be up-regulated. Quantitative measurements provided a molecular signature specific to glycoproteins for determining the degree of plant stress at low temperature. Structural N-glycoproteomics following time-course cold treatments revealed the stress-responsive degradation of high-mannose type N-glycans in ER in response to chilling stress, which may aid in elucidating the cellular mechanisms of protein relocation, transport, trafficking, misfolding and degradation under stress conditions., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Endoplasmic reticulum (ER)-associated degradation (ERAD) is an essential part of an ER-localized protein quality-control system for eliminating terminally misfolded proteins. Recent studies have demonstrated that the ERAD machinery is conserved among yeast, animals, and plants; however, it remains unknown if the plant ERAD system involves plant-specific components. Here we report that the Arabidopsis ethyl methanesulfonate-mutagenized brassinosteroid-insensitive 1 suppressor 7 (EBS7) gene encodes an ER membrane-localized ERAD component that is highly conserved in land plants. Loss-of-function ebs7 mutations prevent ERAD of brassinosteroid insensitive 1-9 (bri1-9) and bri1-5, two ER-retained mutant variants of the cell-surface receptor for brassinosteroids (BRs). As a result, the two mutant receptors accumulate in the ER and consequently leak to the plasma membrane, resulting in the restoration of BR sensitivity and phenotypic suppression of the bri1-9 and bri1-5 mutants. EBS7 accumulates under ER stress, and its mutations lead to hypersensitivity to ER and salt stresses. EBS7 interacts with the ER membrane-anchored ubiquitin ligase Arabidopsis thaliana HMG-CoA reductase degradation 1a (AtHrd1a), one of the central components of the Arabidopsis ERAD machinery, and an ebs7 mutation destabilizes AtHrd1a to reduce polyubiquitination of bri1-9. Taken together, our results uncover a plant-specific component of a plant ERAD pathway and also suggest its likely biochemical function.

Calreticulin (CRT) is a highly conserved chaperone-like lectin that regulates Ca(2+) homeostasis and participates in protein quality control in the endoplasmic reticulum (ER). Most of our CRT knowledge came from mammalian studies, but our understanding of plant CRTs is limited. Many plants contain more than two CRTs that form two distinct groups: CRT1/CRT2 and CRT3. Previous studies on plant CRTs were focused on their Ca(2+)-binding function, but recent studies revealed a crucial role for the Arabidopsis CRT3 in ER retention of a mutant brassinosteroid receptor, brassinosteroid-insensitive 1-9 (bri1-9) and in complete folding of a plant immunity receptor EF-Tu Receptor (EFR). However, little is known about the molecular basis of the functional specification of the CRTs. We have recently shown that the C-terminal domain of CRT3, which is rich in basic residues, is essential for retaining bri1-9 in the ER; however, its role in assisting EFR folding has not been studied. Here, we used an insertional mutant of CRT3, ebs2-8 (EMS mutagenized bri1 suppressor 2-8), in the bri1-9 background as a genetic system to investigate the functional importance of two basic residue clusters in the CRT3's C-terminal domain. Complementation experiments of ebs2-8 bri1-9 with mutant CRT3(M) transgenes showed that a highly conserved basic tetrapeptide Arg(392)Arg (393)Arg(394)Lys(395) is essential but a less conserved basic tetrapeptide Arg(401)Arg(402)Arg(403)Arg(404) is dispensable for the quality control function of CRT3 that retains bri1-9 in the ER and facilitates the complete folding of EFR.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is an integral part of the ER quality-control system that removes toxic misfolded proteins via ubiquitin/proteasome-mediated degradation. Most of our knowledge on ERAD comes from biochemical and genetic studies in yeast and mammalian cells. Although ERAD is known to operate in plant cells, little is known about its molecular components and its biochemical mechanism. A genetic screen for suppressors of the Arabidopsis bri1-9, a weak dwarf mutant caused by ER retention of a structurally defective yet biochemically competent brassinosteroid (BR) receptor BRI1, resulted in identification of the EMS-mutagenized bri1 suppressor 5 (EBS5) gene that encodes an Arabidopsis homolog of the yeast Hrd3/mammalian Sel1L protein known to be involved in ERAD. Loss-of-function ebs5 mutations block the ERAD of bri1-9 and bri1-5, another ER-retained BR receptor. We showed that EBS5 complemented the ERAD defect of the yeast Δhrd3 mutant and interacted with the two mutated BR receptors in plant cells. Using a reverse genetic approach, we discovered that two Arabidopsis homologs of the yeast/mammalian Hrd1, an ER membrane-localized ubiquitin ligase, function redundantly in the ERAD of bri1-9. Together, our results revealed functional roles of two conserved ERAD components in degrading mutated/misfolded receptor-like kinases in Arabidopsis.

Asn-linked glycans, or the glycan code, carry crucial information for protein folding, transport, sorting, and degradation. The biochemical pathway for generating such a code is highly conserved in eukaryotic organisms and consists of ordered assembly of a lipid-linked tetradeccasaccharide. Most of our current knowledge on glycan biosynthesis was obtained from studies of yeast asparagine-linked glycosylation (alg) mutants. By contrast, little is known about biosynthesis and biological functions of N-glycans in plants. Here, we show that loss-of-function mutations in the Arabidopsis thaliana homolog of the yeast ALG12 result in transfer of incompletely assembled glycans to polypeptides. This metabolic defect significantly compromises the endoplasmic reticulum-associated degradation of bri1-9 and bri1-5, two defective transmembrane receptors for brassinosteroids. Consequently, overaccumulated bri1-9 or bri1-5 proteins saturate the quality control systems that retain the two mutated receptors in the endoplasmic reticulum and can thus leak out of the folding compartment, resulting in phenotypic suppression of the two bri1 mutants. Our results strongly suggest that the complete assembly of the lipid-linked glycans is essential for successful quality control of defective glycoproteins in Arabidopsis.

Mammalian calreticulin (CRT) is a multifunctional Ca(2+)-binding protein involved in more than 40 cellular processes in various subcellular compartments, such as Ca(2+) storage and protein folding in the endoplasmic reticulum (ER). CRT homologues were discovered in plants almost 15 years ago, and recent studies revealed that many plant species contain 2 or more CRTs that are members of 2 distinct families, the CRT1/2 family and the plant-specific CRT3 family. However, little is known about their physiological functions. Here we report ebs2 (EMS-mutagenized bri1 suppressor 2) as an allele-specific suppressor of bri1-9, a dwarf Arabidopsis mutant caused by retention of a defective brassinosteroid receptor in the ER. EBS2 encodes the Arabidopsis CRT3 that interacts with ER-localized bri1-9 in a glycan-dependent manner. Loss-of-function ebs2 mutations compromise ER retention of bri1-9 and suppress its dwarfism, whereas EBS2 over-expression enhances its dwarf phenotype. In contrast, mutations of 2 other CRTs or their membrane-localized homologues calnexins had little effect on bri1-9. A domain-swapping experiment revealed that the positively charged C-terminal tail of CRT3 is crucial for its "bri1-9-retainer" function. Our study revealed not only a functional role for a plant-specific CRT, but also functional diversity among the 3 Arabidopsis CRT paralogues.

Endoplasmic reticulum-mediated quality control (ERQC) is a well-studied process in yeast and mammals that retains and disposes misfolded/unassembled polypeptides. By contrast, how plants exert quality control over their secretory proteins is less clear. Here, we report that a mutated brassinosteroid receptor, bri1-5, that carries a Cys69Tyr mutation, is retained in the ER by an overvigilant ERQC system involving three different retention mechanisms. We demonstrate that bri1-5 interacts with two ER chaperones, calnexin and binding protein (BiP), and is degraded by a proteasome-independent endoplasmic reticulum-associated degradation (ERAD). Mutations in components of the calnexin/calreticulin cycle had little effect on the fidelity of the Arabidopsis thaliana ERQC for bri1-5 retention. By contrast, overexpression of bri1-5, treatment with an ERAD inhibitor, RNA interference-mediated BiP silencing, or simultaneous mutations of Cys-69 and its partner Cys-62 can mitigate this quality control, resulting in significant suppression of the bri1-5 phenotype. Thus, bri1-5 is an excellent model protein to investigate plant ERQC/ERAD in a model organism.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a presumed folding sensor of protein quality control in the endoplasmic reticulum (ER). Previous biochemical studies with nonphysiological 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 genetic support for a physiological role of UGGT in high-fidelity ER quality control.

Maintenance of proteome integrity is essential for cell function and survival in changing cellular and environmental conditions. The endoplasmic reticulum (ER) is the major site for the synthesis of secretory and membrane proteins. However, the accumulation of unfolded or misfolded proteins can perturb ER protein homeostasis, leading to ER stress and compromising cellular function. Eukaryotic organisms have evolved sophisticated and conserved protein quality control systems to ensure protein folding fidelity via the unfolded protein response (UPR) and to eliminate potentially harmful proteins via ER-associated degradation (ERAD) and ER-phagy. In this review, we summarize recent advances in our understanding of the mechanisms of ER protein homeostasis in plants and discuss the crosstalk between different quality control systems. Finally, we will address unanswered questions in this field. [ABSTRACT FROM AUTHOR]

Most membrane and secreted proteins are glycosylated on certain asparagine (N) residues in the endoplasmic reticulum (ER), which is crucial for their correct folding and function. Protein folding is a fundamentally inefficient and error-prone process that can be easily interfered by genetic mutations, stochastic cellular events, and environmental stresses. Because misfolded proteins not only lead to functional deficiency but also produce gain-of-function cellular toxicity, eukaryotic organisms have evolved highly conserved ER-mediated protein quality control (ERQC) mechanisms to monitor protein folding, retain and repair incompletely folded or misfolded proteins, or remove terminally misfolded proteins via a unique ER-associated degradation (ERAD) mechanism. A crucial event that terminates futile refolding attempts of a misfolded glycoprotein and diverts it into the ERAD pathway is executed by removal of certain terminal α1,2-mannose (Man) residues of their N -glycans. Earlier studies were centered around an ER-type α1,2-mannosidase that specifically cleaves the terminal α1,2Man residue from the B-branch of the three-branched N-linked Man9GlcNAc2 (GlcNAc for N -acetylglucosamine) glycan, but recent investigations revealed that the signal that marks a terminally misfolded glycoprotein for ERAD is an N -glycan with an exposed α1,6Man residue generated by members of a unique folding-sensitive α1,2-mannosidase family known as ER-degradation enhancing α-mannosidase-like proteins (EDEMs). This review provides a historical recount of major discoveries that led to our current understanding on the role of demannosylating N -glycans in sentencing irreparable misfolded glycoproteins into ERAD. It also discusses conserved and distinct features of the demannosylation processes of the ERAD systems of yeast, mammals, and plants. [ABSTRACT FROM AUTHOR]

To adapt to constantly changing environmental conditions, plants have evolved sophisticated tolerance mechanisms to integrate various stress signals and to coordinate plant growth and development. It is well known that inter-organellar communications play important roles in maintaining cellular homeostasis in response to environmental stresses. The endoplasmic reticulum (ER), extending throughout the cytoplasm of eukaryotic cells, is a central organelle involved in lipid metabolism, Ca2+ homeostasis, and synthesis and folding of secretory and transmembrane proteins crucial to perceive and transduce environmental signals. The ER communicates with the nucleus via the highly conserved unfolded protein response pathway to mitigate ER stress. Importantly, recent studies have revealed that the dynamic ER network physically interacts with other intracellular organelles and endomembrane compartments, such as the Golgi complex, mitochondria, chloroplast, peroxisome, vacuole, and the plasma membrane, through multiple membrane contact sites between closely apposed organelles. In this review, we will discuss the signaling and metabolite exchanges between the ER and other organelles during abiotic stress responses in plants as well as the ER-organelle membrane contact sites and their associated tethering complexes. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum-associated degradation (ERAD) is a highly conserved mechanism to remove misfolded membrane/secretory proteins from the endoplasmic reticulum (ER). While many of the individual components of the ERAD machinery are well characterized in yeast and mammals, our knowledge of a plant ERAD process is rather limited. Here, we report a functional study of an Arabidopsis homolog (AtOS9) of an ER luminal lectin Yos9 (OS-9 in mammals) that recognizes a unique asparagine-linked glycan on misfolded proteins. We discovered that AtOS9 is an ER-localized glycoprotein that is co-expressed with many known/predicted ER chaperones. A T-DNA insertional atos9-t mutation blocks the degradation of a structurally imperfect yet biochemically competent brassinosteroid (BR) receptor bri1-9, causing its increased accumulation in the ER and its consequent leakage to the cell surface responsible for restoring the BR sensitivity and suppressing the dwarfism of the bri1-9 mutant. In addition, we identified a missense mutation in AtOS9 in a recently discovered ERAD mutant ems-mutagenized bri1 suppressor 6 (ebs6-1). Moreover, we showed that atos9-t also inhibits the ERAD of bri1-5, another ER-retained BR receptor, and a misfolded EFR, a BRI1-like receptor for the bacterial translation elongation factor EF-Tu. Furthermore, we found that AtOS9 interacted biochemically and genetically with EBS5, an Arabidopsis homolog of the yeast Hrd3/mammalian Sel1L known to collaborate with Yos9/OS-9 to select ERAD clients. Taken together, our results demonstrated a functional role of AtOS9 in a plant ERAD process that degrades misfolded receptor-like kinases. [ABSTRACT FROM AUTHOR]

Asn-linked glycans, or the glycan code, carry crucial information for protein folding, transport, sorting, and degradation. The biochemical pathway for generating such a code is highly conserved in eukaryotic organisms and consists of ordered assembly of a lipid-linked tetradeccasaccharide. Most of our current knowledge on glycan biosynthesis was obtained from studies of yeast asparagine-linked glycosylation (alg) mutants. By contrast, little is known about biosynthesis and biological functions of N-glycans in plants. Here, we show that loss-of-function mutations in the Arabidopsis thaliana homolog of the yeast ALG12 result in transfer of incompletely assembled glycans to polypeptides. This metabolic defect significantly compromises the endoplasmic reticulum–associated degradation of bri1-9 and bri1-5, two defective transmembrane receptors for brassinosteroids. Consequently, overaccumulated bri1-9 or bri1-5 proteins saturate the quality control systems that retain the two mutated receptors in the endoplasmic reticulum and can thus leak out of the folding compartment, resulting in phenotypic suppression of the two bri1 mutants. Our results strongly suggest that the complete assembly of the lipid-linked glycans is essential for successful quality control of defective glycoproteins in Arabidopsis. [ABSTRACT FROM AUTHOR]

Plants are sensitive to a variety of stresses that cause various diseases throughout their life cycle. However, they have the ability to cope with these stresses using different defense mechanisms. The endoplasmic reticulum (ER) is an important subcellular organelle, primarily recognized as a checkpoint for protein folding. It plays an essential role in ensuring the proper folding and maturation of newly secreted and transmembrane proteins. Different processes are activated when around one-third of newly synthesized proteins enter the ER in the eukaryote cells, such as glycosylation, folding, and/or the assembling of these proteins into protein complexes. However, protein folding in the ER is an error-prone process whereby various stresses easily interfere, leading to the accumulation of unfolded/misfolded proteins and causing ER stress. The unfolded protein response (UPR) is a process that involves sensing ER stress. Many strategies have been developed to reduce ER stress, such as UPR, ER-associated degradation (ERAD), and autophagy. Here, we discuss the ER, ER stress, UPR signaling and various strategies for reducing ER stress in plants. In addition, the UPR signaling in plant development and different stresses have been discussed. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum-associated degradation (ERAD) is a unique mechanism to degrade misfolded proteins via complexes containing several highly-conserved ER-anchored ubiquitin ligases such as HMG-CoA reductase degradation1 (Hrd1). Arabidopsis has a similar Hrd1-containing ERAD machinery; however, our knowledge of this complex is limited. Here we report two closely-related Arabidopsis proteins, Protein Associated With Hrd1-1 (PAWH1) and PAWH2, which share a conserved domain with yeast Altered Inheritance of Mitochondria24. PAWH1 and PAWH2 localize to the ER membrane and associate with Hrd1 via EMS-mutagenized Bri1 Suppressor7 (EBS7), a plant-specific component of the Hrd1 complex. Simultaneously elimination of two PAWHs constitutively activates the unfolded protein response and compromises stress tolerance. Importantly, the pawh1 pawh2 double mutation reduces the protein abundance of EBS7 and Hrd1 and inhibits degradation of several ERAD substrates. Our study not only discovers additional plant-specific components of the Arabidopsis Hrd1 complex but also reveals a distinct mechanism for regulating the Hrd1 stability. Endoplasmic reticulum (ER)-associated degradation (ERAD) removes misfolded proteins from the secretory pathway. Here the authors identify two plant-specific proteins in Arabidopsis, PAWH1 and PAWH2, that bind to and stabilise the ER-anchored ubiquitin ligase Hrd1. [ABSTRACT FROM AUTHOR]