Tandem affinity purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis

Protein ubiquitylation is a central regulatory mechanism that controls numerous processes in plants, including hormone signaling, developmental progression, responses to biotic and abiotic challenges, protein trafficking and chromatin structure. Despite data implicating thousands of plant proteins as targets, so far only a few have been conclusively shown to be ubiquitylated in planta . Here we describe a method to isolate ubiquitin–protein conjugates from Arabidopsis that exploits a stable transgenic line expressing a synthetic poly- UBQ gene encoding ubiquitin (Ub) monomers N-terminally tagged with hexahistidine. Following sequential enrichment by Ub-affinity and nickel chelate-affinity chromatography, the ubiquitylated proteins were trypsinized, separated by two-dimensional liquid chromatography, and analyzed by mass spectrometry. Our list of 54 non-redundant targets, expressed by as many as 90 possible isoforms, included those predicted by genetic studies to be ubiquitylated in plants (EIN3 and JAZ6) or shown to be ubiquitylated in other eukaryotes (ribosomal subunits, elongation factor 1α, histone H1, HSP70 and CDC48), as well as candidates whose control by the Ub/26S proteasome system is not yet appreciated. Ub attachment site(s) were resolved for a subset of these proteins, but surprisingly little sequence consensus was detected, implying that specific residues surrounding the modified lysine are not important determinants for ubiquitylation. We also identified six of the seven available lysine residues on Ub itself as Ub attachment sites, together with evidence for a branched mixed-linkage chain, suggesting that the topologies of Ub chains can be highly complex in plants. Taken together, our method provides a widely applicable strategy to define ubiquitylation in any tissue of intact plants exposed to a wide range of conditions.     

Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co‐expression of affinity‐tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in‐vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non‐ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9‐GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.     

BAK1‐mediated phosphorylation of canonical G protein alpha during flagellin signaling in Arabidopsis

Heterotrimeric G proteins consisting of Gα, Gβ and Gγ are conserved signaling hubs in eukaryotes. Without analogs to canonical animal G protein‐coupled receptors, plant cells are thought to use RGS1 and a yet unknown mechanism to regulate the activity of Gα. Meanwhile, the exact role of canonical Gα in plant innate immunity remains controversial. Here, we report multiple immune deficiencies in the null allele of Arabidopsis Gα (GPA1) in response to bacterial flg22 elicitor, clarifying a positive regulatory role of GPA1 in flg22 signaling. We also detect overall increased phosphorylation of GPA1 but reduced phosphorylation at Thr¹⁹ upon flg22 elicitation. Interestingly, flg22 could not induce phosphorylation of GPA1^T19A and GPA1^T19D, suggesting that the dynamic Thr¹⁹ phosphorylation is required for GPA1 to respond to flg22. Moreover, flg22‐induced GPA1 phosphorylation is largely abolished in the absence of BAK1 in vivo, and BAK1 could phosphorylate GPA1 but not GPA1^T19A in vitro at the phosphorylation sites identified in vivo, suggesting BAK1 is likely the kinase for GPA1 phosphorylation in response to flg22. Furthermore, the T19A mutation could promote flg22‐induced association, rather than dissociation, between GPA1 and RGS1. Taken together, our findings shed new insights into the function and regulation of GPA1 in Arabidopsis defense signaling.     

K27‐linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation

Conjugation of ubiquitin (Ub) to numerous substrate proteins regulates virtually all cellular processes. Eight distinct ubiquitin polymer linkages specifying different functional outcomes are generated in cells. However, the roles of some atypical poly‐ubiquitin topologies, in particular linkages via lysine 27 (K27), remain poorly understood due to a lack of tools for their specific detection and manipulation. Here, we adapted a cell‐based ubiquitin replacement strategy to enable selective and conditional abrogation of K27‐linked ubiquitylation, revealing that this ubiquitin linkage type is essential for proliferation of human cells. We demonstrate that K27‐linked ubiquitylation is predominantly a nuclear modification whose ablation deregulates nuclear ubiquitylation dynamics and impairs cell cycle progression in an epistatic manner with inactivation of the ATPase p97/VCP. Moreover, we show that a p97‐proteasome pathway model substrate (Ub(G76V)‐GFP) is directly modified by K27‐linked ubiquitylation, and that disabling the formation of K27‐linked ubiquitin signals or blocking their decoding via overexpression of the K27 linkage‐specific binder UCHL3 impedes Ub(G76V)‐GFP turnover at the level of p97 function. Our findings suggest a critical role of K27‐linked ubiquitylation in supporting cell fitness by facilitating p97‐dependent processing of ubiquitylated nuclear proteins.     

Neurite outgrowth in PC12 cells. Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (>/=2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub approximately E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub approximately E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of 125 I-labeled beta-lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3). However, Ub-dependent proteolysis of 125I-labeled beta-lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Ub-protein conjugates. Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta-lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Ub-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala. These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.     

ESCRT-I Mediates FLS2 Endosomal Sorting and Plant Immunity

The plant immune receptor FLAGELLIN SENSING 2 (FLS2) is present at the plasma membrane and is internalized following activation of its ligand flagellin (flg22). We show that ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT (ESCRT)-I subunits play roles in FLS2 endocytosis in Arabidopsis. VPS37-1 co-localizes with FLS2 at endosomes and immunoprecipitates with the receptor upon flg22 elicitation. Vps37-1 mutants are reduced in flg22-induced FLS2 endosomes but not in endosomes labeled by Rab5 GTPases suggesting a defect in FLS2 trafficking rather than formation of endosomes. FLS2 localizes to the lumen of multivesicular bodies, but this is altered in vps37-1 mutants indicating compromised endosomal sorting of FLS2 by ESCRT-I loss-of-function. VPS37-1 and VPS28-2 are critical for immunity against bacterial infection through a role in stomatal closure. Our findings identify that VPS37-1, and likewise VPS28-2, regulate late FLS2 endosomal sorting and reveals that ESCRT-I is critical for flg22-activated stomatal defenses involved in plant immunity.     

Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation

Although several proteasome subunits have been shown to bind ubiquitin (Ub) chains, many ubiquitylated substrates also associate with 26S proteasomes via “shuttling factors.” Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a Ub-associated domain; therefore, it is unclear how they bind Ub. Here, we show that deletion of Ddi2 leads to the accumulation of Ub conjugates with K11/K48 branched chains. We found using affinity copurifications that Ddi2 binds Ub conjugates through its Ub-like domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding Ub conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of Ub conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking the endoprotease activity of Ddi2 either genetically or with the HIV protease inhibitor nelfinavir increased its binding to Ub conjugates but decreased its binding to proteasomes and reduced subsequent protein degradation by proteasomes leading to further accumulation of Ub conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum–associated protein degradation and delivers K11/K48-ubiquitylated proteins to the proteasome. We conclude that the protease activity of Ddi2 influences this shuttling factor activity, promotes protein turnover, and helps prevent endoplasmic reticulum stress, which may explain nelfinavir’s ability to enhance cell killing by proteasome inhibitors.     

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies

Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington’s disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.     

Biochemical and genetic requirements for function of the immune response regulator BOTRYTIS-INDUCED KINASE1 in plant growth, ethylene signaling, and PAMP-triggered immunity in Arabidopsis

Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) regulates immune responses to a distinct class of pathogens. Here, mechanisms underlying BIK1 function and its interactions with other immune response regulators were determined. We describe BIK1 function as a component of ethylene (ET) signaling and PAMP-triggered immunity (PTI) to fungal pathogens. BIK1 in vivo kinase activity increases in response to flagellin peptide (flg22) and the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) but is blocked by inhibition of ET perception. BIK1 induction by flg22, ACC, and pathogens is strictly dependent on EIN3, and the bik1 mutation results in altered expression of ET-regulated genes. BIK1 site-directed mutants were used to determine residues essential for phosphorylation and biological functions in planta, including PTI, ET signaling, and plant growth. Genetic analysis revealed flg22-induced PTI to Botrytis cinerea requires BIK1, EIN2, and HUB1 but not genes involved in salicylate (SA) functions. BIK1-mediated PTI to Pseudomonas syringae is modulated by SA, ET, and jasmonate signaling. The coi1 mutation suppressed several bik1 phenotypes, suggesting that COI1 may act as a repressor of BIK1 function. Thus, common and distinct mechanisms underlying BIK1 function in mediating responses to distinct pathogens are uncovered. In sum, the critical role of BIK1 in plant immune responses hinges upon phosphorylation, its function in ET signaling, and complex interactions with other immune response regulators.     

A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles

Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single-step immunoenrichment of ubiquitylated peptides with peptide fractionation and high-resolution mass spectrometry to investigate endogenous ubiquitylation sites. We precisely map 11,054 endogenous putative ubiquitylation sites (diglycine-modified lysines) on 4,273 human proteins. The presented data set covers 67% of the known ubiquitylation sites and contains 10,254 novel sites on proteins with diverse cellular functions including cell signaling, receptor endocytosis, DNA replication, DNA damage repair, and cell cycle progression. Our method enables site-specific quantification of ubiquitylation in response to cellular perturbations and is applicable to any cell type or tissue. Global quantification of ubiquitylation in cells treated with the proteasome inhibitor MG-132 discovers sites that are involved in proteasomal degradation, and suggests a nonproteasomal function for almost half of all sites. Surprisingly, ubiquitylation of about 15% of sites decreased more than twofold within four hours of MG-132 treatment, showing that inhibition of proteasomal function can dramatically reduce ubiquitylation on many sites with non-proteasomal functions. Comparison of ubiquitylation sites with acetylation sites reveals an extensive overlap between the lysine residues targeted by these two modifications. However, the crosstalk between these two post-translational modifications is significantly less frequent on sites that show increased ubiquitylation upon proteasome inhibition. Taken together, we report the largest site-specific ubiquitylation dataset in human cells, and for the first time demonstrate proteome-wide, site-specific quantification of endogenous putative ubiquitylation sites.     

Loss of Arabidopsis thaliana Dynamin-Related Protein 2B Reveals Separation of Innate Immune Signaling Pathways

Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC ⁻, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca²⁺-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC⁻. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.     

Regulating the Regulators: Recent Revelations in the Control of E3 Ubiquitin Ligases

Since its discovery as a post-translational signal for protein degradation, our understanding of ubiquitin (Ub) has vastly evolved. Today, we recognize that the role of Ub signaling is expansive and encompasses diverse processes including cell division, the DNA damage response, cellular immune signaling, and even organismal development. With such a wide range of functions comes a wide range of regulatory mechanisms that control the activity of the ubiquitylation machinery. Ub attachment to substrates occurs through the sequential action of three classes of enzymes, E1s, E2s, and E3s. In humans, there are 2 E1s, ∼35 E2s, and hundreds of E3s that work to attach Ub to thousands of cellular substrates. Regulation of ubiquitylation can occur at each stage of the stepwise Ub transfer process, and substrates can also impact their own modification. Recent studies have revealed elegant mechanisms that have evolved to control the activity of the enzymes involved. In this minireview, we highlight recent discoveries that define some of the various mechanisms by which the activities of E3-Ub ligases are regulated.     

MultiDsk: A Ubiquitin-Specific Affinity Resin

Ubiquitylation is a highly diverse and complex post-translational modification for the regulation of protein function and stability. Studies of ubiquitylation have, however, been hampered by its rapid reversal in cell extracts, for example through the action of de-ubiquitylating enzymes (DUBs). Here we describe a novel ubiquitin-binding protein reagent, MultiDsk, composed of an array of five UBA domains from the yeast ubiquitin-binding protein Dsk2, fused to GST. MultiDsk binds ubiquitylated substrates with unprecedented avidity, and can be used as both an affinity resin to study protein ubiquitylation, and to effectively protect ubiquitylated proteins from the action of DUBs and the proteasome in crude cell extracts. We use the resin to show that the Def1 protein becomes ubiquitylated in response to DNA damage, and to isolate ubiquitylated forms of RNA polymerase II.     

Analysis of flagellin perception mediated by flg22 receptor OsFLS2 in rice

Plants have sensitive perception systems that recognize various pathogen-derived molecules. We previously reported that rice detects flagellin from a rice-incompatible strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, which induces subsequent immune responses involving cell death. The mechanism of flagellin perception in rice, however, has remained obscure. In this study, we found that flg22, a peptide derived from the flagellin N-terminus, induced weak immune responses without cell death in cultured rice cells. To elucidate the mechanism by which flg22 induced signaling in rice, we characterized OsFLS2, the rice ortholog of AtFLS2, which mediates flg22 perception. Heterologous expression of OsFLS2 functions in Arabidopsis, showing the conservation of the flg22 signaling pathway across divergent plant taxa. OsFLS2-overexpressing rice cultured cells generated stronger immune responses with the induction of cell death following stimulation with flg22 and flagellin. However, examination of the growth rate of the compatible strain in inoculated OsFLS2-overexpressing rice could not confirm bacterial growth suppression compared with wild-type rice. These results suggest that rice possesses a conserved flagellin perception system utilizing the FLS2 receptor which, when upregulated, hardly affects resistance against compatible A. avenae.     

PAMP (Pathogen-associated Molecular Pattern)-induced Changes in Plasma Membrane Compartmentalization Reveal Novel Components of Plant Immunity

Plasma membrane compartmentalization spatiotemporally regulates cell-autonomous immune signaling in animal cells. To elucidate immediate early protein dynamics at the plant plasma membrane in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin (flg22) we employed quantitative mass spectrometric analysis on detergent-resistant membranes (DRMs) of Arabidopsis thaliana suspension cells. This approach revealed rapid and profound changes in DRM protein composition following PAMP treatment, prominently affecting proton ATPases and receptor-like kinases, including the flagellin receptor FLS2. We employed reverse genetics to address a potential contribution of a subset of these proteins in flg22-triggered cellular responses. Mutants of three candidates (DET3, AHA1, FER) exhibited a conspicuous defect in the PAMP-triggered accumulation of reactive oxygen species. In addition, these mutants showed altered mitogen-activated protein kinase (MAPK) activation, a defect in PAMP-triggered stomatal closure as well as altered bacterial infection phenotypes, which revealed three novel players in elicitor-dependent oxidative burst control and innate immunity. Our data provide evidence for dynamic elicitor-induced changes in the membrane compartmentalization of PAMP signaling components.     

The interplay between MAMP and SA signaling

There are two major modes for plant recognition of biotrophic microbial pathogens. In one mode, plant pattern recognition receptors (PRRs) recognize microbe associated molecular patterns (MAMPs, also called PAMPs), which are molecules such as flg22, a fragment of bacterial flagellin. In the other mode, the products of plant resistance (R) genes recognize pathogen effectors or host proteins modified by effectors. Salicylic acid (SA) -mediated defense responses are an important part of R gene-mediated resistance. It was not clear how these two signaling mechanisms interact with each other. Recently, we reported that treatment with flg22 triggered SA accumulation in Arabidopsis leaves. Disruptions of SA signaling components strongly affected MAMP-triggered gene expression responses. Flg22-triggered resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) was partly dependent on SA signaling. Our results demonstrated the importance of SA signaling in flg22-triggered resistance and, at the same time, the importance of some other signaling mechanism(s) in this resistance. Here we discuss potential signaling components of flg22-triggered SA accumulation and other signaling mechanisms potentially contributing to flg22-triggered resistance to Pst DC3000.Key words: arabidopsis, expression profiling, MAMP, PAD4, PAMP, salicylic acid (SA), SID2     

DYNAMIN-RELATED PROTEIN DRP1A functions with DRP2B in plant growth,flg22-immune responses,and endocytosis

Ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (FLS2) is critical for maintaining its proper abundance in the plasma membrane (PM) to initiate and subsequently down regulate cellular immune responses to bacterial flagellin or flg22-peptide. The molecular components governing PM abundance of FLS2, however, remain mostly unknown. Here, we identified Arabidopsis (Arabidopsis thaliana) DYNAMIN-RELATED PROTEIN1A (DRP1A), a member of a plant-specific family of large dynamin GTPases, as a critical contributor to ligand-induced endocytosis of FLS2 and its physiological roles in flg22-signaling and immunity against Pseudomonas syringae pv. tomato DC3000 bacteria in leaves. Notably, drp1a single mutants displayed similar flg22-defects as those previously reported for mutants in another dynamin-related protein, DRP2B, that was previously shown to colocalize with DRP1A. Our study also uncovered synergistic roles of DRP1A and DRP2B in plant growth and development as drp1a drp2b double mutants exhibited severely stunted roots and cotyledons, as well as defective cell shape, cytokinesis, and seedling lethality. Furthermore, drp1a drp2b double mutants hyperaccumulated FLS2 in the PM prior to flg22-treatment and exhibited a block in ligand-induced endocytosis of FLS2, indicating combinatorial roles for DRP1A and DRP1B in governing PM abundance of FLS2. However, the increased steady-state PM accumulation of FLS2 in drp1a drp2b double mutants did not result in increased flg22 responses. We propose that DRP1A and DRP2B are important for the regulation of PM-associated levels of FLS2 necessary to attain signaling competency to initiate distinct flg22 responses, potentially through modulating the lipid environment in defined PM domains.

A plant-specific large dynamin GTPase is required for plant responses against bacterial pathogens and, with another dynamin, regulates the cell surface composition for plant growth and defense.