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The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization.

Steffens A, Bräutigam A, Jakoby M, Hülskamp M - PLoS Biol. (2015)

Bottom Line: Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi.We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs.Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation) interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

View Article: PubMed Central - PubMed

Affiliation: Botanical Institute, Biocenter, Cologne University, Cologne, Germany.

ABSTRACT
Members of the highly conserved class of BEACH domain containing proteins (BDCPs) have been established as broad facilitators of protein-protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI) is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1), associates to mRNA processing bodies (P-bodies), and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation) interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

No MeSH data available.


Related in: MedlinePlus

The SPI protein interacts with DCP1.(A) Schematic presentation of the domain organization of the SPI protein: the ARMADILLO repeats (ARM), the Concanavalin A-like lectin domain (ConA), and the C-terminal PBW module (SPI-PBW). (B) Yeast two-hybrid interactions. Upper part: double transformed yeast cells on selective dropout medium lacking leucine (-L) and tryptophan (-W). Bottom part: interaction between SPI-PBW, N-terminally fused to the GAL4 Binding Domain (BD), and DCP1 and other P-body core components fused to the GAL4 Activation Domain (AD), on selective dropout medium lacking leucine (-L), tryptophan (-W), and histidine (-H), supplemented with 5 mM 3-Aminotrizole (3AT). The Green Fluorescent Protein (GFP), N-terminally fused to the GAL4-AD, has been included as negative control. (C) Coprecipitation of SPI-PBW-His6 with DCP1-MBP. Throughputs (TP), wash fractions (WF), and resin-bound MBP fusions (B) were detected by α-MBP (upper part) and α-His6 (lower part) antibody staining. GST-SPI-PBW-His6 (~110 kDa, arrowhead) coprecipitated with MBP-DCP1 (~ 83 kDa), but not with MBP (~42 kDa). Samples detected on different blots are separated by lines. (D) FRETE (in %) was measured in whole leaf epidermis cells (whole cells) and stationary P-bodies (PBs). YFP was bleached in whole cells (for details see Materials and Methods). Mean FRETE’s for 35Spro:YFP-gSPI and 35Spro:DCP1-CFP (n = 11 cells) or 35Spro:YFP and 35Spro:DCP1-CFP (n = 10 cells) are shown. Error bars represent standard deviations for whole cells, and the standard deviation of the mean for PBs (n = 31 stationary PBs derived from whole cell samples). Two-tailed student’s t test was performed to compare FRETE between 35Spro:YFP-gSPI/35Spro:DCP1-CFP and 35Spro:YFP/35Spro:DCP1-CFP for each group (*** p < 0.001). (E) Representative images of 35Spro:DCP1-CFP in a transiently transfected leaf epidermis cell prior to (left) and after (middle) Acceptor-photobleaching (AP). For a better visualization, the increase of fluorescence intensity of DCP1-CFP after AP is presented in pseudocolors (right), see color scale for comparison. A group of stationary PBs is highlighted by the boxed area and magnified (lower row). Yellow arrowheads in magnifications mark stationary PBs used for FRET quantifications. Scale bars: 30 μm.
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pbio.1002188.g001: The SPI protein interacts with DCP1.(A) Schematic presentation of the domain organization of the SPI protein: the ARMADILLO repeats (ARM), the Concanavalin A-like lectin domain (ConA), and the C-terminal PBW module (SPI-PBW). (B) Yeast two-hybrid interactions. Upper part: double transformed yeast cells on selective dropout medium lacking leucine (-L) and tryptophan (-W). Bottom part: interaction between SPI-PBW, N-terminally fused to the GAL4 Binding Domain (BD), and DCP1 and other P-body core components fused to the GAL4 Activation Domain (AD), on selective dropout medium lacking leucine (-L), tryptophan (-W), and histidine (-H), supplemented with 5 mM 3-Aminotrizole (3AT). The Green Fluorescent Protein (GFP), N-terminally fused to the GAL4-AD, has been included as negative control. (C) Coprecipitation of SPI-PBW-His6 with DCP1-MBP. Throughputs (TP), wash fractions (WF), and resin-bound MBP fusions (B) were detected by α-MBP (upper part) and α-His6 (lower part) antibody staining. GST-SPI-PBW-His6 (~110 kDa, arrowhead) coprecipitated with MBP-DCP1 (~ 83 kDa), but not with MBP (~42 kDa). Samples detected on different blots are separated by lines. (D) FRETE (in %) was measured in whole leaf epidermis cells (whole cells) and stationary P-bodies (PBs). YFP was bleached in whole cells (for details see Materials and Methods). Mean FRETE’s for 35Spro:YFP-gSPI and 35Spro:DCP1-CFP (n = 11 cells) or 35Spro:YFP and 35Spro:DCP1-CFP (n = 10 cells) are shown. Error bars represent standard deviations for whole cells, and the standard deviation of the mean for PBs (n = 31 stationary PBs derived from whole cell samples). Two-tailed student’s t test was performed to compare FRETE between 35Spro:YFP-gSPI/35Spro:DCP1-CFP and 35Spro:YFP/35Spro:DCP1-CFP for each group (*** p < 0.001). (E) Representative images of 35Spro:DCP1-CFP in a transiently transfected leaf epidermis cell prior to (left) and after (middle) Acceptor-photobleaching (AP). For a better visualization, the increase of fluorescence intensity of DCP1-CFP after AP is presented in pseudocolors (right), see color scale for comparison. A group of stationary PBs is highlighted by the boxed area and magnified (lower row). Yellow arrowheads in magnifications mark stationary PBs used for FRET quantifications. Scale bars: 30 μm.

Mentions: Consistent with a role for BDCPs in membrane trafficking and dynamics, several studies identified membrane-associated proteins as binding partners of BDCPs [26–30]. To identify interactors of plant BDCPs, we performed yeast two-hybrid cDNA library screens using the C-terminal fragment of SPI containing its PBW domain module (referred to as SPI-PBW hereafter, Fig 1A) as bait. Surprisingly, we identified the evolutionarily conserved P-body core component DCP1 as an interaction partner (Fig 1B). All other tested decapping complex proteins including DCP2, DCP5, or VCS did not show interactions with SPI in yeast two-hybrid assays. The interaction of SPI-PBW and DCP1 was confirmed in pull-down experiments with bacterially expressed proteins. Gluthatione S-Transferase (GST)/His6-fusions of SPI-PBW were efficiently bound to resins labeled with Maltose Binding Protein (MBP)-tagged DCP1, while no binding was detected with the negative control MBP alone (Fig 1C). To analyze the interaction between full-length SPI and DCP1 in Arabidopsis leaf epidermis cells, we performed Förster-Resonance Energy Transfer (FRET)-Acceptor Photobleaching (AP) experiments (Fig 1D and 1E). We expressed YFP-tagged full-length genomic SPI (acceptor) and DCP1-CFP (cyan fluorescent protein; donor) under the 35S promoter and measured their FRET efficiencies (FRETE). We measured FRETE of about 27% in whole cells, indicating that the interaction between DCP1 and SPI occurs in vivo. To test whether the interaction takes place at P-bodies, we analyzed the fraction of immobile P-bodies [31]. Here, the FRETE was 23% (Fig 1E), indicating that SPI and DCP1 interact at P-bodies. No significant FRET was detected between DCP1-CFP and free YFP (yellow fluorescent protein) as a negative control. Donor emissions of cells transfected with DCP1-CFP alone were used as a photobleaching corrective (Fig 1D). The intracellular localization of the SPI-PBW/DCP1 interaction was independently analyzed by Bimolecular Fluorescence Complementation (BiFC) assays in transiently transformed Nicotiana benthamiana leaf epidermis cells. Like shown for full-length SPI and DCP1 in FRET-AP assays, we found SPI-PBW and DCP1 interacting at distinct cytoplasmic dot-like structures. These completely colocalized with DCP2-mCHERRY (mCHERRY is a monomeric mutant of DsRED) (Fig 2A). To exclude that the presence of another P-body component influences the interaction behavior of SPI-PBW and DCP1, we confirmed our observations in BiFC assays coexpressing free RFP (red fluorescent protein) instead of DCP2-mCHERRY (Fig 2B).


The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization.

Steffens A, Bräutigam A, Jakoby M, Hülskamp M - PLoS Biol. (2015)

The SPI protein interacts with DCP1.(A) Schematic presentation of the domain organization of the SPI protein: the ARMADILLO repeats (ARM), the Concanavalin A-like lectin domain (ConA), and the C-terminal PBW module (SPI-PBW). (B) Yeast two-hybrid interactions. Upper part: double transformed yeast cells on selective dropout medium lacking leucine (-L) and tryptophan (-W). Bottom part: interaction between SPI-PBW, N-terminally fused to the GAL4 Binding Domain (BD), and DCP1 and other P-body core components fused to the GAL4 Activation Domain (AD), on selective dropout medium lacking leucine (-L), tryptophan (-W), and histidine (-H), supplemented with 5 mM 3-Aminotrizole (3AT). The Green Fluorescent Protein (GFP), N-terminally fused to the GAL4-AD, has been included as negative control. (C) Coprecipitation of SPI-PBW-His6 with DCP1-MBP. Throughputs (TP), wash fractions (WF), and resin-bound MBP fusions (B) were detected by α-MBP (upper part) and α-His6 (lower part) antibody staining. GST-SPI-PBW-His6 (~110 kDa, arrowhead) coprecipitated with MBP-DCP1 (~ 83 kDa), but not with MBP (~42 kDa). Samples detected on different blots are separated by lines. (D) FRETE (in %) was measured in whole leaf epidermis cells (whole cells) and stationary P-bodies (PBs). YFP was bleached in whole cells (for details see Materials and Methods). Mean FRETE’s for 35Spro:YFP-gSPI and 35Spro:DCP1-CFP (n = 11 cells) or 35Spro:YFP and 35Spro:DCP1-CFP (n = 10 cells) are shown. Error bars represent standard deviations for whole cells, and the standard deviation of the mean for PBs (n = 31 stationary PBs derived from whole cell samples). Two-tailed student’s t test was performed to compare FRETE between 35Spro:YFP-gSPI/35Spro:DCP1-CFP and 35Spro:YFP/35Spro:DCP1-CFP for each group (*** p < 0.001). (E) Representative images of 35Spro:DCP1-CFP in a transiently transfected leaf epidermis cell prior to (left) and after (middle) Acceptor-photobleaching (AP). For a better visualization, the increase of fluorescence intensity of DCP1-CFP after AP is presented in pseudocolors (right), see color scale for comparison. A group of stationary PBs is highlighted by the boxed area and magnified (lower row). Yellow arrowheads in magnifications mark stationary PBs used for FRET quantifications. Scale bars: 30 μm.
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pbio.1002188.g001: The SPI protein interacts with DCP1.(A) Schematic presentation of the domain organization of the SPI protein: the ARMADILLO repeats (ARM), the Concanavalin A-like lectin domain (ConA), and the C-terminal PBW module (SPI-PBW). (B) Yeast two-hybrid interactions. Upper part: double transformed yeast cells on selective dropout medium lacking leucine (-L) and tryptophan (-W). Bottom part: interaction between SPI-PBW, N-terminally fused to the GAL4 Binding Domain (BD), and DCP1 and other P-body core components fused to the GAL4 Activation Domain (AD), on selective dropout medium lacking leucine (-L), tryptophan (-W), and histidine (-H), supplemented with 5 mM 3-Aminotrizole (3AT). The Green Fluorescent Protein (GFP), N-terminally fused to the GAL4-AD, has been included as negative control. (C) Coprecipitation of SPI-PBW-His6 with DCP1-MBP. Throughputs (TP), wash fractions (WF), and resin-bound MBP fusions (B) were detected by α-MBP (upper part) and α-His6 (lower part) antibody staining. GST-SPI-PBW-His6 (~110 kDa, arrowhead) coprecipitated with MBP-DCP1 (~ 83 kDa), but not with MBP (~42 kDa). Samples detected on different blots are separated by lines. (D) FRETE (in %) was measured in whole leaf epidermis cells (whole cells) and stationary P-bodies (PBs). YFP was bleached in whole cells (for details see Materials and Methods). Mean FRETE’s for 35Spro:YFP-gSPI and 35Spro:DCP1-CFP (n = 11 cells) or 35Spro:YFP and 35Spro:DCP1-CFP (n = 10 cells) are shown. Error bars represent standard deviations for whole cells, and the standard deviation of the mean for PBs (n = 31 stationary PBs derived from whole cell samples). Two-tailed student’s t test was performed to compare FRETE between 35Spro:YFP-gSPI/35Spro:DCP1-CFP and 35Spro:YFP/35Spro:DCP1-CFP for each group (*** p < 0.001). (E) Representative images of 35Spro:DCP1-CFP in a transiently transfected leaf epidermis cell prior to (left) and after (middle) Acceptor-photobleaching (AP). For a better visualization, the increase of fluorescence intensity of DCP1-CFP after AP is presented in pseudocolors (right), see color scale for comparison. A group of stationary PBs is highlighted by the boxed area and magnified (lower row). Yellow arrowheads in magnifications mark stationary PBs used for FRET quantifications. Scale bars: 30 μm.
Mentions: Consistent with a role for BDCPs in membrane trafficking and dynamics, several studies identified membrane-associated proteins as binding partners of BDCPs [26–30]. To identify interactors of plant BDCPs, we performed yeast two-hybrid cDNA library screens using the C-terminal fragment of SPI containing its PBW domain module (referred to as SPI-PBW hereafter, Fig 1A) as bait. Surprisingly, we identified the evolutionarily conserved P-body core component DCP1 as an interaction partner (Fig 1B). All other tested decapping complex proteins including DCP2, DCP5, or VCS did not show interactions with SPI in yeast two-hybrid assays. The interaction of SPI-PBW and DCP1 was confirmed in pull-down experiments with bacterially expressed proteins. Gluthatione S-Transferase (GST)/His6-fusions of SPI-PBW were efficiently bound to resins labeled with Maltose Binding Protein (MBP)-tagged DCP1, while no binding was detected with the negative control MBP alone (Fig 1C). To analyze the interaction between full-length SPI and DCP1 in Arabidopsis leaf epidermis cells, we performed Förster-Resonance Energy Transfer (FRET)-Acceptor Photobleaching (AP) experiments (Fig 1D and 1E). We expressed YFP-tagged full-length genomic SPI (acceptor) and DCP1-CFP (cyan fluorescent protein; donor) under the 35S promoter and measured their FRET efficiencies (FRETE). We measured FRETE of about 27% in whole cells, indicating that the interaction between DCP1 and SPI occurs in vivo. To test whether the interaction takes place at P-bodies, we analyzed the fraction of immobile P-bodies [31]. Here, the FRETE was 23% (Fig 1E), indicating that SPI and DCP1 interact at P-bodies. No significant FRET was detected between DCP1-CFP and free YFP (yellow fluorescent protein) as a negative control. Donor emissions of cells transfected with DCP1-CFP alone were used as a photobleaching corrective (Fig 1D). The intracellular localization of the SPI-PBW/DCP1 interaction was independently analyzed by Bimolecular Fluorescence Complementation (BiFC) assays in transiently transformed Nicotiana benthamiana leaf epidermis cells. Like shown for full-length SPI and DCP1 in FRET-AP assays, we found SPI-PBW and DCP1 interacting at distinct cytoplasmic dot-like structures. These completely colocalized with DCP2-mCHERRY (mCHERRY is a monomeric mutant of DsRED) (Fig 2A). To exclude that the presence of another P-body component influences the interaction behavior of SPI-PBW and DCP1, we confirmed our observations in BiFC assays coexpressing free RFP (red fluorescent protein) instead of DCP2-mCHERRY (Fig 2B).

Bottom Line: Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi.We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs.Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation) interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

View Article: PubMed Central - PubMed

Affiliation: Botanical Institute, Biocenter, Cologne University, Cologne, Germany.

ABSTRACT
Members of the highly conserved class of BEACH domain containing proteins (BDCPs) have been established as broad facilitators of protein-protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI) is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1), associates to mRNA processing bodies (P-bodies), and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation) interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

No MeSH data available.


Related in: MedlinePlus