Cak1 Is Required for Kin28 Phosphorylation and Activation In Vivo

Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.     

In Planta Assessment of the Role of Thioredoxin h Proteins in the Regulation of S-Locus Receptor Kinase Signaling in Transgenic Arabidopsis

The self-incompatibility (SI) response of the Brassicaceae is mediated by allele-specific interaction between the stigma-localized S-locus receptor kinase (SRK) and its ligand, the pollen coat-localized S-locus cysteine-rich protein (SCR). Based on work in Brassica spp., the thioredoxin h-like proteins THL1 and THL2, which interact with SRK, have been proposed to function as oxidoreductases that negatively regulate SRK catalytic activity. By preventing the spontaneous activation of SRK in the absence of SCR ligand, these thioredoxins are thought to be essential for the success of cross pollinations in self-incompatible plants. However, the in planta role of thioredoxins in the regulation of SI signaling has not been conclusively demonstrated. Here, we addressed this issue using Arabidopsis thaliana plants transformed with the SRKb-SCRb gene pair isolated from self-incompatible Arabidopsis lyrata. These plants express an intense SI response, allowing us to exploit the extensive tools and resources available in A. thaliana for analysis of SI signaling. To test the hypothesis that SRK is redox regulated by thioredoxin h, we expressed a mutant form of SRKb lacking a transmembrane-localized cysteine residue thought to be essential for the SRK-thioredoxin h interaction. We also analyzed transfer DNA insertion mutants in the A. thaliana orthologs of THL1 and THL2. In neither case did we observe an effect on the pollination responses of SRKb-expressing stigmas toward incompatible or compatible pollen. Our results are consistent with the conclusion that, contrary to their proposed role, thioredoxin h proteins are not required to prevent the spontaneous activation of SRK in the A. thaliana stigma.Many flowering plants possess self-incompatibility (SI), a genetic system that promotes outcrossing by preventing self-fertilization. In the Brassicaceae family, the SI response is controlled by haplotypes of the S locus, each of which contains two genes that encode highly polymorphic proteins, the S-locus receptor kinase (SRK), which is a plasma membrane resident single-pass transmembrane Ser/Thr receptor kinase displayed at the surface of stigma epidermal cells (Stein et al., 1991; Takasaki et al., 2000), and the S-locus Cys-rich protein (SCR), which is the pollen coat-localized ligand for SRK (Schopfer et al., 1999; Kachroo et al., 2001; Takayama et al., 2001). SRK and SCR exhibit allele-specific interactions, whereby only SRK and SCR encoded by the same S-locus haplotype interact. In a self-pollination, the binding of this “self” pollen-borne SCR to the extracellular domain of SRK activates the SRK kinase, thereby triggering a cellular response in stigma epidermal cells that causes inhibition of pollen germination and tube penetration into the stigma epidermal cell wall (for review, see Tantikanjana et al., 2010).Tight regulation of SRK kinase activity and its signaling cascade is critical for productive pollen-stigma interactions because constitutive (i.e. SCR-independent) activity of the receptor is expected to result in sterile stigmas that reject both compatible and incompatible pollen. In the classical view of ligand-activated receptor kinases, the receptor occurs as catalytically inactive monomers in the absence of ligand and only becomes activated upon ligand-induced dimerization (for review, see Lemmon and Schlessinger, 2010). However, some receptor kinases in both animals (Chan et al., 2000; Ehrlich et al., 2011) and plants (Giranton et al., 2000; Wang et al., 2005, 2008; Shimizu et al., 2010; Bücherl et al., 2013) form catalytically inactive dimers or oligomers in the absence of ligand, with receptor activation presumably resulting from ligand-induced higher order oligomerization or conformational changes (Lemmon and Schlessinger, 2010). Similar to the latter receptors, SRK forms oligomers in unpollinated stigmas, i.e. in the absence of SCR (Giranton et al., 2000), at least partly via ligand-independent dimerization domains located within the SRK extracellular domain (Naithani et al., 2007). It has been proposed that these ligand-independent SRK oligomers are maintained in an inactive state by thioredoxins, the ubiquitous small oxidoreductases that reduce disulfide bridges in proteins (Buchanan and Balmer, 2005). This hypothesis is supported by the following observations: (1) two Brassica napus thioredoxins, the Thioredoxin H-Like proteins THL1 and THL2, were identified as SRK interactors in a yeast (Saccharomyces cerevisiae) two-hybrid screen that used the B. napus SRK₉₁₀ kinase domain as bait (Bower et al., 1996); (2) when purified from pistils or insect cells, the Brassica oleracea SRK₃ variant was found to exhibit constitutive autophosphorylation activity in vitro, and this activity was inhibited by Escherichia coli-expressed THL1 proteins and was restored by addition of pollen coat proteins containing self but not of pollen coat proteins containing nonself SCR (Cabrillac et al., 2001); (3) the catalytic activity of THL1 was required for its inhibition of SRK₃ autophosphorylation activity in vitro (Cabrillac et al., 2001); and (4) antisense suppression of THL1/THL2 gene expression in the stigmas of a self-compatible B. napus strain reportedly produced a low-level constitutive incompatibility (Haffani et al., 2004), as might be expected if the THL1/THL2 proteins prevent the spontaneous activation of SRK-mediated signaling in stigmas.These observations notwithstanding, the in planta role of thioredoxin h proteins as negative regulators of SRK activity has not been conclusively demonstrated. To date, this proposed function has only been evaluated in a self-compatible strain of B. napus (Haffani et al., 2004). Consequently, it is not known if the proposed inhibitory effect of these thioredoxins on SRK catalytic activity is manifested in self-incompatible stigmas and if it applies to all SRK variants, be they derived from Brassica spp. or other self-incompatible species of the Brassicaceae such as Arabidopsis lyrata.In this study, we tested the in planta role of thioredoxin h proteins in the regulation of SI signaling using a transgenic self-incompatible Arabidopsis thaliana model that we generated by transforming A. thaliana with the SRKb-SCRb gene pair isolated from the Sb haplotype of self-incompatible A. lyrata (Kusaba et al., 2001; Nasrallah et al., 2002, 2004). We had previously shown that the stigmas of A. thaliana SRKb-SCRb transformants can exhibit an SI response that is as robust as the SI response observed in naturally self-incompatible A. lyrata, demonstrating that A. thaliana, which harbors nonfunctional S-locus haplotypes (Kusaba et al., 2001; Sherman-Broyles et al., 2007; Shimizu et al., 2008; Boggs et al., 2009c), has nevertheless retained all other factors required for SI. In view of the availability in A. thaliana of a highly efficient transformation method and numerous genetic resources, the A. thaliana SRK-SCR transgenic model has enabled the use of experimental approaches that are difficult or impossible to implement in Brassica species and has thus proven to be an invaluable platform for in planta analysis of SRK and SI signaling (Liu et al., 2007; Boggs et al., 2009a, 2009b; Tantikanjana et al., 2009; Tantikanjana and Nasrallah, 2012).We therefore used this transgenic A. thaliana self-incompatible model to determine if abolishing the proposed SRK-thioredoxin h interaction or eliminating expression of the major thioredoxin h proteins expressed in stigmas would affect the outcome of self- or cross pollination. To this end, we expressed a mutant form of SRKb that lacked the Cys residue previously shown to be required for the interaction of SRK with THLs (Mazzurco et al., 2001), and we analyzed plants carrying knockout insertional mutations in thioredoxin h genes. Our results are inconsistent with the proposed role of thioredoxin h proteins as negative regulators of SRK catalytic activity and SI signaling.     

Ouabain Releases Striatal Polyamines In Vivo Independently of N-Methyl-d-Aspartate Receptor Activation

Abstract: Intrastriatally infused ouabain (200 or 1,000 μ M ) markedly increased the extracellular levels of striatal spermidine and spermine in dialysis experiments in halothane-anesthetized rats. The effects of ouabain (1 m M ) on sper- midine release were rapid and unaffected by local infusion of the competitive N -methyl- d -aspartate (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)propyl-1 -phosphonic acid (CPP; 100 μ M ) or by systemically administered MK-801 (0.3 mg/kg i.p.), both of which treatments markedly inhibit the effects of intrastriatally administered NMDA. The peak effects of ouabain (1 m M ) on spermine release were delayed with respect to those on spermidine release, or to the effects of NMDA, and were also insensitive to locally administered CPP (100 μ M ). However, systemically administered MK-801 (0.3 mg/kg i.p., 30 min before the striatal infusion of drugs), which totally inhibits the effects of NMDA, or CPP (10 mg/kg i.p.; 30 min before the striatal infusion of drugs) partially inhibited the effects of ouabain on spermine release, suggesting partial mediation of the delayed effects of ouabain on spermine release by indirect NMDA-receptor activation. Despite partial sensitivity of ouabain-induced spermine release to systemically administered NMDA antagonists, both spermidine and spermine can be released in vivo by sodium-pump inhibition, independently of NMDA-receptor activation.     

A Mutant of Arabidopsis thaliana Which Lacks Activation of RuBP Carboxylase In Vivo

A mutant of Arabidopsis thaliana has been isolated in which ribulose-1,5-bisphosphate carboxylase is present in a nonactivatable form in vivo. The mutation appears to affect carboxylase activation specifically, and not any other enzyme of the photosynthesis or photorespiratory cycles. The effect of the mutation on carboxylase activation is indirect, inasmuch as the properties of ribulose-1,5-bisphosphate carboxylase purified from the mutant are not distinguishable from those of the wild type enzyme. The mutant requires high levels of atmospheric CO₂ for growth because photosynthesis is severely impaired in atmospheres containing normal levels of CO₂, irrespective of the atmospheric O₂ concentration. In this respect, the mutant is distinguished from previously described high-CO₂ requiring mutants of Arabidopsis which have defects in photorespiratory carbon or nitrogen metabolism.     

Differential In Vivo Regulation of Steroid Hormone Receptor Activation by Cdc37p

The CDC37 gene is essential for the activity of p60^v-src when expressed in yeast cells. Since the activation pathway for p60^v-src and steroid hormone receptors is similar, the present study analyzed the hormone-dependent transactivation by androgen receptors and glucocorticoid receptors in yeast cells expressing a mutant version of the CDC37 gene. In this mutant, hormone-dependent transactivation by androgen receptors was defective at both permissive and restrictive temperatures, although transactivation by glucocorticoid receptors was mildly defective only at the restrictive temperature. Cdc37p appears to function via the androgen receptor ligand-binding domain, although it does not influence receptor hormone-binding affinity. Models for Cdc37p regulation of steroid hormone receptors are discussed.     

Independent S-Locus Mutations Caused Self-Fertility in Arabidopsis thaliana

A common yet poorly understood evolutionary transition among flowering plants is a switch from outbreeding to an inbreeding mode of mating. The model plant Arabidopsis thaliana evolved to an inbreeding state through the loss of self-incompatibility, a pollen-rejection system in which pollen recognition by the stigma is determined by tightly linked and co-evolving alleles of the S-locus receptor kinase (SRK) and its S-locus cysteine-rich ligand (SCR). Transformation of A. thaliana, with a functional AlSRKb-SCRb gene pair from its outcrossing relative A. lyrata, demonstrated that A. thaliana accessions harbor different sets of cryptic self-fertility–promoting mutations, not only in S-locus genes, but also in other loci required for self-incompatibility. However, it is still not known how many times and in what manner the switch to self-fertility occurred in the A. thaliana lineage. Here, we report on our identification of four accessions that are reverted to full self-incompatibility by transformation with AlSRKb-SCRb, bringing to five the number of accessions in which self-fertility is due to, and was likely caused by, S-locus inactivation. Analysis of S-haplotype organization reveals that inter-haplotypic recombination events, rearrangements, and deletions have restructured the S locus and its genes in these accessions. We also perform a Quantitative Trait Loci (QTL) analysis to identify modifier loci associated with self-fertility in the Col-0 reference accession, which cannot be reverted to full self-incompatibility. Our results indicate that the transition to inbreeding occurred by at least two, and possibly more, independent S-locus mutations, and identify a novel unstable modifier locus that contributes to self-fertility in Col-0.     

Identification of Two Tyrosine Residues Required for the Intramolecular Mechanism Implicated in GIT1 Activation

GIT1 is an ArfGAP and scaffolding protein regulating cell adhesion and migration. The multidomain structure of GIT1 allows the interaction with several partners. Binding of GIT1 to some of its partners requires activation of the GIT1 polypeptide. Our previous studies indicated that binding of paxillin to GIT1 is enhanced by release of an intramolecular interaction between the amino-terminal and carboxy-terminal portions that keeps the protein in a binding-incompetent state. Here we have addressed the mechanism mediating this intramolecular inhibitory mechanism by testing the effects of the mutation of several formerly identified GIT1 phosphorylation sites on the binding to paxillin. We have identified two tyrosines at positions 246 and 293 of the human GIT1 polypeptide that are needed to keep the protein in the inactive conformation. Interestingly, mutation of these residues to phenylalanine did not affect binding to paxillin, while mutation to either alanine or glutamic acid enhanced binding to paxillin, without affecting the constitutive binding to the Rac/Cdc42 exchange factor βPIX. The involvement of the two tyrosine residues in the intramolecular interaction was supported by reconstitution experiments showing that these residues are important for the binding between the amino-terminal fragment and carboxy-terminal portions of GIT1. Either GIT1 or GIT1-N tyrosine phosphorylation by Src and pervanadate treatment to inhibit protein tyrosine phosphatases did not affect the intramolecular binding between the amino- and carboxy-terminal fragments, nor the binding of GIT1 to paxillin. Mutations increasing the binding of GIT1 to paxillin positively affected cell motility, measured both by transwell migration and wound healing assays. Altogether these results show that tyrosines 246 and 293 of GIT1 are required for the intramolecular inhibitory mechanism that prevents the binding of GIT1 to paxillin. The data also suggest that tyrosine phosphorylation may not be sufficient to release the intramolecular interaction that keeps GIT1 in the inactive conformation.     

Crucial Positively Charged Residues for Ligand Activation of the GPR35 Receptor

GPR35 is a G protein-coupled receptor expressed in the immune, gastrointestinal, and nervous systems in gastric carcinomas and is implicated in heart failure and pain perception. We investigated residues in GPR35 responsible for ligand activation and the receptor structure in the active state. GPR35 contains numerous positively charged amino acids that face into the binding pocket that cluster in two distinct receptor regions, TMH3-4-5-6 and TMH1-2-7. Computer modeling implicated TMH3-4-5-6 for activation by the GPR35 agonists zaprinast and pamoic acid. Mutation results for the TMH1-2-7 region of GPR35 showed no change in ligand efficacies at the K1.32A, R2.65A, R7.33A, and K7.40A mutants. However, mutation of arginine residues in the TMH3-4-5-6 region (R4.60, R6.58, R3.36, R(164), and R(167) in the EC2 loop) had effects on signaling for one or both agonists tested. R4.60A resulted in a total ablation of agonist-induced activation in both the β-arrestin trafficking and ERK1/2 activation assays. R6.58A increased the potency of zaprinast 30-fold in the pERK assay. The R(167)A mutant decreased the potency of pamoic acid in the β-arrestin trafficking assay. The R(164)A and R(164)L mutants decreased potencies of both agonists. Similar trends for R6.58A and R(167)A were observed in calcium responses. Computer modeling showed that the R6.58A mutant has additional interactions with zaprinast. R3.36A did not express on the cell surface but was trapped in the cytoplasm. The lack of surface expression of R3.36A was rescued by a GPR35 antagonist, CID2745687. These results clearly show that R4.60, R(164), R(167), and R6.58 play crucial roles in the agonist initiated activation of GPR35.     

Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin

Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.     

Selective Release of Spermine and Spermidine from the Rat Striatum by N-Methyl-d-Aspartate Receptor Activation In Vivo

The intrastriatal infusion of N-methyl-D-aspartate (NMDA; 250-1,000 microM) via a dialysis cannula in anesthetized rats resulted in a marked and rapid increase in the concentrations of spermine and spermidine recovered in the dialysate. Extracellular concentrations of NMDA-released spermine and spermidine were calculated to be in the low micromolar range. Putrescine levels were not significantly affected by NMDA. The effects of NMDA (500 microM) were blocked by the previous systemic injection of MK-801 (3 mg/kg, i.p.) but were insensitive to the intrastriatal infusion of tetrodotoxin (1 microM). Intrastriatally infused kainate or quisqualate (1,000 microM) did not increase polyamine levels in the dialysate. Spermine and spermidine dialysate levels were also significantly increased by the infusion of high concentrations of K+ (greater than 100 mM), although the effects of K+ were considerably less marked than those of NMDA. Striatal polyamines are released into the extracellular space specifically by NMDA receptor activation. Because of their multiple effects on receptor- and voltage-operated cation channels, polyamines that are released by NMDA receptor activation may play an important role in phenomena already attributed to NMDA receptor stimulation, such as long-term potentiation, synaptic plasticity, and neurotoxicity.     

Activation of Notch Signaling Is Required for Cholangiocarcinoma Progression and Is Enhanced by Inactivation of p53 In Vivo

Cholangiocacinoma (CC) is a cancer disease with rising incidence. Notch signaling has been shown to be deregulated in many cancers. However, the role of this signaling pathway in the carcinogenesis of CC is still not fully explored. In this study, we investigated the effects of Notch inhibition by γ-secretase inhibitor IX (GSI IX) in cultured human CC cell lines and we established a transgenic mouse model with liver specific expression of the intracellular domain of Notch (Notch-ICD) and inactivation of tumor suppressor p53. GSI IX treatment effectively impaired cell proliferation, migration, invasion, epithelial to mesenchymal transition and growth of softagar colonies. In vivo overexpression of Notch-ICD together with an inactivation of p53 significantly increased tumor burden and showed CC characteristics. Conclusion: Our study highlights the importance of Notch signaling in the tumorigenesis of CC and demonstrates that additional inactivation of p53 in vivo.     

Domains of a Transit Sequence Required for in Vivo Import in Arabidopsis Chloroplasts

Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.     

Transient Receptor Potential Ankyrin 1 Receptor Activation In Vitro and In Vivo by Pro-tussive Agents: GRC 17536 as a Promising Anti-Tussive Therapeutic

Cough is a protective reflex action that helps clear the respiratory tract which is continuously exposed to airborne environmental irritants. However, chronic cough presents itself as a disease in its own right and despite its global occurrence; the molecular mechanisms responsible for cough are not completely understood. Transient receptor potential ankyrin1 (TRPA1) is robustly expressed in the neuronal as well as non-neuronal cells of the respiratory tract and is a sensor of a wide range of environmental irritants. It is fast getting acceptance as a key biological sensor of a variety of pro-tussive agents often implicated in miscellaneous chronic cough conditions. In the present study, we demonstrate in vitro direct functional activation of TRPA1 receptor by citric acid which is routinely used to evoke cough in preclinical and clinical studies. We also show for the first time that a potent and selective TRPA1 antagonist GRC 17536 inhibits citric acid induced cellular Ca⁺² influx in TRPA1 expressing cells and the citric acid induced cough response in guinea pigs. Hence our data provides a mechanistic link between TRPA1 receptor activation in vitro and cough response induced in vivo by citric acid. Furthermore, we also show evidence for TRPA1 activation in vitro by the TLR4, TLR7 and TLR8 ligands which are implicated in bacterial/respiratory virus pathogenesis often resulting in chronic cough. In conclusion, this study highlights the potential utility of TRPA1 antagonist such as GRC 17536 in the treatment of miscellaneous chronic cough conditions arising due to diverse causes but commonly driven via TRPA1.     

Peroxisomes Are Required for in Vivo Nitric Oxide Accumulation in the Cytosol following Salinity Stress of Arabidopsis Plants

Peroxisomes are unique organelles involved in multiple cellular metabolic pathways. Nitric oxide (NO) is a free radical active in many physiological functions under normal and stress conditions. Using Arabidopsis (Arabidopsis thaliana) wild type and mutants expressing green fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo, this study analyzes the temporal and cell distribution of NO during the development of 3-, 5-, 8-, and 11-d-old Arabidopsis seedlings and shows that Arabidopsis peroxisomes accumulate NO in vivo. Pharmacological analyses using nitric oxide synthase (NOS) inhibitors detected the presence of putative calcium-dependent NOS activity. Furthermore, peroxins Pex12 and Pex13 appear to be involved in transporting the putative NOS protein to peroxisomes, since pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, registered lower NO content. Additionally, we show that under salinity stress (100 mm NaCl), peroxisomes are required for NO accumulation in the cytosol, thereby participating in the generation of peroxynitrite (ONOO⁻) and in increasing protein tyrosine nitration, which is a marker of nitrosative stress.Peroxisomes are single membrane-bound organelles whose basic enzymatic constituents are catalase and H₂O₂-producing flavin oxidases as their basic enzymatic and are found in virtually all eukaryotic cell types (Corpas et al., 2001; Hayashi and Nishimura, 2006; Reumann et al., 2007; Pracharoenwattana and Smith, 2008; Palma et al., 2009). These oxidative organelles are characterized by metabolic plasticity, as their enzymatic content can vary according to the organism, cell/tissue type, and environmental conditions (Mullen et al., 2001; Hayashi and Nishimura, 2003; Corpas et al., 2009a). In higher plants, peroxisomes contain a complex battery of antioxidative enzymes, such as catalase, superoxide dismutase, the components of the ascorbate-glutathione cycle, and the NADP-dehydrogenases of the pentose-P pathway (Corpas et al., 2009a). The generation of superoxide radicals has also been reported in the matrices and membranes of peroxisomes (López-Huertas et al., 1999; del Río et al., 2006). All these findings point to the important role played by peroxisomes in the cellular metabolism of reactive oxygen species (Corpas et al., 2001, 2009a; del Río et al., 2006).Nitric oxide (NO) is a free radical involved in many physiological functions under normal and stress conditions in both animal and plant cells (Arasimowicz and Floryszak-Wieczorek, 2007; Corpas et al., 2007a, 2008; Neill et al., 2008). Unlike animal systems, knowledge of NO generation and subcellular location in plants remains largely elusive, and the data are sometimes contradictory and ambiguous (Zemojtel et al., 2006; Jasid et al., 2006; Gas et al., 2009). In previous studies, we detected l-Arg-dependent nitric oxide synthase (NOS) activity in isolated pea (Pisum sativum) leaf peroxisomes (Barroso et al., 1999). In a later study, using electron paramagnetic resonance techniques, we demonstrated the presence of NO in these types of peroxisomes (Corpas et al., 2004). However, several issues, such as whether NO is released into the cytosol and the physiological function of this free radical, remain unresolved.In this study, we provide an in vivo demonstration that Arabidopsis peroxisomes are essential for NO accumulation in the cytosol, thus participating in the generation of nitrosative stress under salinity conditions. In addition, using Arabidopsis mutants pex12 and pex13, we also suggest that these peroxins are involved in importing into peroxisomes the enzyme responsible for NO generation.     

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

A growing body of evidence suggests that the extracellular domain of the epithelial Na⁺ channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na⁺ self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ_E455 is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β_E446A generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na⁺ self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.