Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis

Abscisic acid (ABA) is a key plant hormone involved in diverse physiological and developmental processes, including abiotic stress responses and the regulation of stomatal aperture and seed germination. Abscisic acid glucosyl ester (ABA-GE) is a hydrolyzable ABA conjugate that accumulates in the vacuole and presumably also in the endoplasmic reticulum. Deconjugation of ABA-GE by the endoplasmic reticulum and vacuolar β-glucosidases allows the rapid formation of free ABA in response to abiotic stress conditions such as dehydration and salt stress. ABA-GE further contributes to the maintenance of ABA homeostasis, as it is the major ABA catabolite exported from the cytosol. In this work, we identified that the import of ABA-GE into vacuoles isolated from Arabidopsis (Arabidopsis thaliana) mesophyll cells is mediated by two distinct membrane transport mechanisms: proton gradient-driven and ATP-binding cassette (ABC) transporters. Both systems have similar K_m values of approximately 1 mm. According to our estimations, this low affinity appears nevertheless to be sufficient for the continuous vacuolar sequestration of ABA-GE produced in the cytosol. We further demonstrate that two tested multispecific vacuolar ABCC-type ABC transporters from Arabidopsis exhibit ABA-GE transport activity when expressed in yeast (Saccharomyces cerevisiae), which also supports the involvement of ABC transporters in ABA-GE uptake. Our findings suggest that the vacuolar ABA-GE uptake is not mediated by specific, but rather by several, possibly multispecific, transporters that are involved in the general vacuolar sequestration of conjugated metabolites.Abscisic acid (ABA) is a major plant hormone involved in various physiological and developmental processes. ABA signaling is fundamental in plant responses to abiotic stresses, including drought, cold, osmotic, and salt stress (Cutler et al., 2010). The best-characterized function of ABA is the regulation of stomatal aperture in response to environmental signals, such as soil and air humidity, temperature, and CO₂ concentration (Nilson and Assmann, 2007; Kim et al., 2010). However, ABA also has important functions in seed development, dormancy, and germination (Holdsworth et al., 2008), lateral root formation (Galvan-Ampudia and Testerink, 2011), and leaf senescence (Lim et al., 2007). Besides, ABA is not restricted only to plants; it was also identified to have functions in species from all kingdoms, including humans, and may even have universal functions (e.g. in UV-B stress response; Tossi et al., 2012).ABA is synthesized de novo from the carotenoid zeaxanthin, whereby the first ABA-specific biosynthetic step occurs in the plastid and the final two steps take place in the cytosol (Nambara and Marion-Poll, 2005). The catabolism of ABA is mediated via oxidative and Glc conjugation pathways (Nambara and Marion-Poll, 2005). The ABA 8′-hydroxylation catalyzed by P450 cytochromes of the CYP707A subfamily represents the predominant catabolic pathway of ABA and has been demonstrated to be a key regulatory step in ABA action (Kushiro et al., 2004). The major oxidative ABA catabolites, phaseic acid (PA) and dihydroxyphaseic acid (DPA), exhibit lower and no biological activity, respectively (Sharkey and Raschke, 1980; Kepka et al., 2011). The conjugation of ABA and its oxidative catabolites PA and DPA with Glc catalyzed by UDP-glucosyltransferases represents the other mechanism of ABA inactivation. Abscisic acid glucosyl ester (ABA-GE) appears to be the major conjugate, which was found in various organs of different plant species (Piotrowska and Bajguz, 2011). In contrast to the oxidative pathway, the inactivation of ABA by Glc conjugation is reversible, and hydrolysis of ABA-GE catalyzed by β-glucosidases results in free ABA (Dietz et al., 2000; Lee et al., 2006; Xu et al., 2012). ABA-GE levels were shown to substantially increase during dehydration and specific seed developmental and germination stages (Boyer and Zeevaart, 1982; Hocher et al., 1991; Chiwocha et al., 2003). Furthermore, ABA-GE is present in the xylem sap, where it was shown to increase under drought, salt, and osmotic stress (Sauter et al., 2002). Apoplastic ABA β-glucosidases in leaves have been suggested to mediate the release of free ABA from xylem-borne ABA-GE (Dietz et al., 2000). Therefore, ABA-GE was proposed to be a root-to-shoot signaling molecule. However, under drought stress, ABA-mediated stomatal closure occurs independently of root ABA biosynthesis (Christmann et al., 2007). Thus, the involvement of ABA-GE in root-to-shoot signaling of water stress conditions remains to be revealed (Goodger and Schachtman, 2010).The intracellular compartmentalization of ABA and its catabolites is important for ABA homeostasis (Xu et al., 2013). Free ABA, PA, and DPA mainly occur in the extravacuolar compartments. In contrast to these oxidative ABA catabolites, ABA-GE has been reported to accumulate in vacuoles (Bray and Zeevaart, 1985; Lehmann and Glund, 1986). Since the sequestered ABA-GE can instantaneously provide ABA via a one-step hydrolysis, this conjugate and its compartmentalization may be of importance in the maintenance of ABA homeostasis. The identification of the endoplasmic reticulum (ER)-localized β-glucosidase AtBG1 that specifically hydrolyzes ABA-GE suggests that ABA-GE is also present in the ER (Lee et al., 2006). Plants lacking functional AtBG1 exhibit pronounced ABA-deficiency phenotypes, including sensitivity to dehydration, impaired stomatal closure, earlier germination, and lower ABA levels. Hydrolysis of ER-localized ABA-GE, therefore, represents an alternative pathway for the generation of free cytosolic ABA (Lee et al., 2006; Bauer et al., 2013). This finding raised the question of whether vacuolar ABA-GE also has an important function as an ABA reservoir. This hypothesis was supported by recent identifications of two vacuolar β-glucosidases that hydrolyze vacuolar ABA-GE (Wang et al., 2011; Xu et al., 2013). The vacuolar AtBG1 homolog AtBG2 forms high molecular weight complexes, which are present at low levels under normal conditions but significantly accumulate under dehydration stress. AtBG2 knockout plants displayed a similar, although less pronounced, phenotype to AtBG1 mutants: elevated sensitivity to drought and salt stress, while overexpression of AtBG2 resulted in exactly the opposite effect (i.e. increased drought tolerance). The other identified vacuolar ABA-GE glucosidase, BGLU10, exhibits comparable mutant phenotypes to AtBG2 (Wang et al., 2011). This redundancy may explain the less pronounced mutant phenotypes of vacuolar ABA-GE glucosidases compared with the ER-localized AtBG1. Moreover, the fact that overexpression of the vacuolar AtBG2 is able to phenotypically complement AtBG1 deletion mutants indicates an important role of vacuolar ABA-GE as a pool for free ABA during the abiotic stress response (Xu et al., 2012).The described accumulation and functions of vacuolar ABA-GE raise the question of by which mechanisms ABA-GE is sequestered into the vacuoles. To answer this question, we synthesized radiolabeled ABA-GE and characterized the ABA-GE transport into isolated mesophyll vacuoles. We showed that the vacuole comprises two distinct transport systems involved in the accumulation of ABA-GE: proton gradient-dependent and directly energized ATP-binding cassette (ABC)-type transport. In a targeted approach, we furthermore show that the Arabidopsis (Arabidopsis thaliana) ABC transporters AtABCC1 and AtABCC2 exhibit ABA-GE transport activity in vitro.     

Targeted Ubiquitination and Degradation of G-Protein-Coupled Receptor Kinase 5 by the DDB1-CUL4 Ubiquitin Ligase Complex

The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors (GPCRs) and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA-binding protein 1 (DDB1), an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex-dependent proteolysis pathway.     

Structural Basis of the Binding of Merlin FERM Domain to the E3 Ubiquitin Ligase Substrate Adaptor DCAF1

The tumor suppressor gene Nf2 product, Merlin, plays vital roles in controlling proper development of organ sizes by specifically binding to a large number of target proteins localized both in cytoplasm and nuclei. The FERM domain of Merlin is chiefly responsible for its binding to target proteins, although the molecular basis governing these interactions are poorly understood due to lack of structural information. Here, we report the crystal structure of the Merlin FERM domain in complex with its binding domain derived from the E3 ubiquitin ligase substrate adaptor DCAF1 (also known as VPRBP). Unlike target binding modes found in ERM proteins, the Merlin-FERM binding domain of DCAF1 folds as a β-hairpin and binds to the α1/β5-groove of the F3 lobe of Merlin-FERM via extensive hydrophobic interactions. In addition to providing the first structural glimpse of a Merlin-FERM·target complex, the structure of the Merlin·DCAF1 complex is likely to be valuable for understanding the interactions of Merlin with its binding partners other than DCAF1.     

The E3 Ubiquitin Ligase HOS1 Regulates Arabidopsis Flowering by Mediating CONSTANS Degradation Under Cold Stress

The timing of flowering is coordinated by a web of gene regulatory networks that integrates developmental and environmental cues in plants. Light and temperature are two major environmental determinants that regulate flowering time. Although prolonged treatment with low nonfreezing temperatures accelerates flowering by stable repression of FLOWERING LOCUS C (FLC), repeated brief cold treatments delay flowering. Here, we report that intermittent cold treatments trigger the degradation of CONSTANS (CO), a central activator of photoperiodic flowering; daily treatments caused suppression of the floral integrator FLOWERING LOCUS T (FT) and delayed flowering. Cold-induced CO degradation is mediated via a ubiquitin/proteasome pathway that involves the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1). HOS1-mediated CO degradation occurs independently of the well established cold response pathways. It is also independent of the light signaling repressor CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase and light wavelengths. CO has been shown to play a key role in photoperiodic flowering. Here, we demonstrated that CO served as a molecular hub, integrating photoperiodic and cold stress signals into the flowering genetic pathways. We propose that the HOS1-CO module contributes to the fine-tuning of photoperiodic flowering under short term temperature fluctuations, which often occur during local weather disturbances.     

Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1^K29R-K31R-GFP remained. The HPG1-1 (Rsp5^P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.     

The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O₂. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O₂-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.     

Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) functions as an E3 ubiquitin ligase and mediates a variety of developmental processes in Arabidopsis by targeting a number of key regulators for ubiquitination and degradation. Here, we identify a novel COP1 interacting protein, COP1 SUPPRESSOR 2 (CSU2). Loss of function mutations in CSU2 suppress the constitutive photomorphogenic phenotype of cop1-6 in darkness. CSU2 directly interacts with COP1 via their coiled-coil domains and is recruited by COP1 into nuclear speckles in living plant cells. Furthermore, CSU2 inhibits COP1 E3 ubiquitin ligase activity in vitro, and represses COP1 mediated turnover of HY5 in cell-free extracts. We propose that in csu2 cop1-6 mutants, the lack of CSU2’s repression of COP1 allows the low level of COP1 to exhibit higher activity that is sufficient to prevent accumulation of HY5 in the dark, thus restoring the etiolated phenotype. In addition, CSU2 is required for primary root development under normal light growth condition.     

IRT1 DEGRADATION FACTOR1, a RING E3 Ubiquitin Ligase,Regulates the Degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis

Fe is an essential micronutrient for plant growth and development; plants have developed sophisticated strategies to acquire ferric Fe from the soil. Nongraminaceous plants acquire Fe by a reduction-based mechanism, and graminaceous plants use a chelation-based mechanism. In Arabidopsis thaliana, which uses the reduction-based method, IRON-REGULATED TRANSPORTER1 (IRT1) functions as the most important transporter for ferrous Fe uptake. Rapid and constitutive degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. IRT1 degradation involves ubiquitination. To identify the specific E3 ubiquitin ligases involved in IRT1 degradation, we screened a set of insertional mutants in RING-type E3 ligases and identified a mutant that showed delayed degradation of IRT1 and loss of IRT1-ubiquitin complexes. The corresponding gene was designated IRT1 DEGRADATION FACTOR1 (IDF1). Evidence of direct interaction between IDF1 and IRT1 in the plasma membrane supported the role of IDF1 in IRT1 degradation. IRT1 accumulation was reduced when coexpressed with IDF1 in yeast or Xenopus laevis oocytes. IDF1 function was RING domain dependent. The idf1 mutants showed increased tolerance to Fe deficiency, resulting from increased IRT1 levels. This evidence indicates that IDF1 directly regulates IRT1 degradation through its RING-type E3 ligase activity.     

Ubiquitination and Degradation of the Hominoid-Specific Oncoprotein TBC1D3 Is Mediated by CUL7 E3 Ligase

Expression of the hominoid-specific TBC1D3 oncoprotein enhances growth factor receptor signaling and subsequently promotes cellular proliferation and survival. Here we report that TBC1D3 is degraded in response to growth factor signaling, suggesting that TBC1D3 expression is regulated by a growth factor-driven negative feedback loop. To gain a better understanding of how TBC1D3 is regulated, we studied the effects of growth factor receptor signaling on TBC1D3 post-translational processing and turnover. Using a yeast two-hybrid screen, we identified CUL7, the scaffolding subunit of the CUL7 E3 ligase complex, as a TBC1D3-interacting protein. We show that CUL7 E3 ligase ubiquitinates TBC1D3 in response to serum stimulation. Moreover, TBC1D3 recruits F-box 8 (Fbw8), the substrate recognition domain of CUL7 E3 ligase, in pull-down experiments and in an in vitro assay. Importantly, alkaline phosphatase treatment of TBC1D3 suppresses its ability to recruit Fbw8, indicating that TBC1D3 phosphorylation is critical for its ubiquitination and degradation. We conclude that serum- and growth factor-stimulated TBC1D3 ubiquitination and degradation are regulated by its interaction with CUL7-Fbw8.     

Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase

Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD.