Dataset Information

Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3.

ABSTRACT: The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.

SUBMITTER: Demir F

PROVIDER: S-EPMC3657796 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3.

Demir Fatih F Horntrich Claudia C Blachutzik Jörg O JO Scherzer Sönke S Reinders Yvonne Y Kierszniowska Sylwia S Schulze Waltraud X WX Harms Gregory S GS Hedrich Rainer R Geiger Dietmar D Kreuzer Ines I

Proceedings of the National Academy of Sciences of the United States of America 20130429 20

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cel ...[more]

PMID: 23630285

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Project description:Two datasets: StREM1.3: methyl-beta cyclodextrin-dependent distribution of StREM1.3 to detergent resistant and detergent soluble membrane fractions. SLAH3: localization of SLAH3 to detergent resistant and detergent soluble fractions dependent on co-expression with CPK21 and ABI1. Data analysis: Protein identiﬁcation and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a ﬁxed modiﬁcation; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modiﬁcations. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identiﬁed by MaxQuant were excluded from further analys Protein identiﬁcation and ion intensity quantitation was carried out by MaxQuant version 1.3.0.5. Spectra were matched against the Arabidopsis proteome (TAIR10, 35,386 entries) using Andromeda. Thereby, carbamidomethylation of cysteine was set as a ﬁxed modiﬁcation; oxidation of methionine as well as phosphorylation of serine, threonine, and tyrosine was set as variable modiﬁcations. Because label-free quantitation based on ion intensities was used, multiplicity was set to 1. Mass tolerance for the database search was set to 20 ppm on full scans and 0.5 Da for fragment ions. Retention time matching between runs was chosen within a time window of 2 min. Peptide false discovery rate (FDR), protein FDR was set to 0.01, and site FDR was set to 0.05. Contaminants and reverse hits identiﬁed by MaxQuant were excluded from further analysis. PXD000211 is also assoicated with the same study/paper.

Project description:Analysis of Arabidopsis nanodomains shows that Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channnel SLAH3. The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin AtRem 1.3. After transient coexpression, CPK21 interacted with SLAH3 (SLAC1 homolog 3) and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21. Data processing and bioinformatics: MS result peak files were run on a Mascot daemon using the Mascot algorithm (version 2.2; Matrix Science) with TAIR v9 protein database, trypsin/P as protease. Allowed fixed modification was carbamidomethylation (C), and variable modifications were oxidized methionines (N) and pyroglutamic acid (pyro-Glu at N-terminal Q). Peptide and fragment mass tolerance were set to +-1.5 Da for the ion trap and +-0.2 Da for the Quad-TOF, maximum missed cleavages to two, and only singly, doubly, and triply charged ions were analyzed. After manual inspection, protein identifications with at least three unique identified peptides were automatically approved. Protein identifications based on two unique identified were only regarded if the two unique identified peptides displayed an ions score above the Mascot significance threshold for P < 0.05 (Mascot score >40 for the ion trap and >32 for the QUAD-TOF). Detailed information for the identified proteins was queried from the Uniprot consortium database (www.uniprot.org) and The Arabidopsis Information Center (TAIR: www.arabidopsis.org). The functional classification was assigned manually according to the annotations found in these two databases. Two independent biological replicates were analyzed: first setup of DRM isolation was performed with the detergents TritonX-100 and Brij-98 in-solution and in-gel digested. The second setup for DRM isolation was only performed with the detergent Triton X-100 and in-gel digested but subjected to methylcyclodextrin treatment to identify sterol-dependent lipid nanodomain proteins. For the proteomic analysis of DRMs data sets from both experimental setups were analyzed; the MCD treatment was only analyzed for the second setup. Protein amounts were estimated only for the MCD treatment using the Exponentially Modified Protein Abundance Index, which correlates with the number of observed and observable peptides per protein.

Project description:Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene expression. Here, we assessed the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors on the nuclear envelope mediate effects on cardiomyocyte gene expression. Immunoblots of nucleus-enriched fractions from isolated cardiomyocytes indicated the presence of AT1R and AT2R proteins that copurified with the nuclear membrane marker nucleoporin-62 and histone-3, but not markers of plasma (calpactin-I), Golgi (GRP-78), or endoplasmic reticulum (GM130) membranes. Confocal microscopy revealed AT1R and AT2R proteins on nuclear membranes. Microinjected Ang-II preferentially bound to nuclear sites of isolated cardiomyocytes. AT1R and AT2R ligands enhanced de novo RNA synthesis in isolated cardiomyocyte nuclei incubated with [alpha-(32)P]UTP (e.g. 36.0 +/- 6.0 cpm/ng of DNA control versus 246.4 +/- 15.4 cpm/ng of DNA Ang-II, 390.1 +/- 15.5 cpm/ng of DNA L-162313 (AT1), 180.9 +/- 7.2 cpm/ng of DNA CGP42112A (AT2), p < 0.001). Ang-II application to cardiomyocyte nuclei enhanced NFkappaB mRNA expression, a response that was suppressed by co-administration of AT1R (valsartan) and/or AT2R (PD123177) blockers. Dose-response experiments with Ang-II applied to purified cardiomyocyte nuclei versus intact cardiomyocytes showed greater increases in NFkappaB mRNA levels at saturating concentrations with approximately 2-fold greater affinity upon nuclear application, suggesting preferential nuclear signaling. AT1R, but not AT2R, stimulation increased [Ca(2+)] in isolated cardiomyocyte nuclei. Inositol 1,4,5-trisphosphate receptor blockade by 2-aminoethoxydiphenyl borate prevented AT1R-mediated Ca(2+) release and attenuated AT1R-mediated transcription initiation responses. We conclude that cardiomyocyte nuclear membranes possess angiotensin receptors that couple to nuclear signaling pathways and regulate transcription. Signaling within the nuclear envelope (e.g. from intracellularly synthesized Ang-II) may play a role in Ang-II-mediated changes in cardiac gene expression, with potentially important mechanistic and therapeutic implications.

Dataset Information

Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3.

Publications

Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3.

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