Limits...
Grain setting defect1 (GSD1) function in rice depends on S-acylation and interacts with actin 1 (OsACT1) at its C-terminal.

Gui J, Zheng S, Shen J, Li L - Front Plant Sci (2015)

Bottom Line: Association with the PM is mediated by S-acylation of cysteine residues Cys-524 and Cys-527, in a sequence of 45 amino acid residues essential for GSD1 function in rice.Furthermore, the coiled-coil domain in GSD1 is necessary for sufficient interaction with OsACT1.Together, these results reveal that GSD1 attaches to the PM through S-acylation and interacts with OsACT1 through its coiled-coil domain structure to regulate plasmodesmata conductance for photoassimilate transport in rice.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Shanghai, China.

ABSTRACT
Grain setting defect1 (GSD1), a plant-specific remorin protein specifically localized at the plasma membrane (PM) and plasmodesmata of phloem companion cells, affects grain setting in rice through regulating the transport of photoassimilates. Here, we show new evidence demonstrating that GSD1 is localized at the cytoplasmic face of the PM and a stretch of 45 amino acid residues at its C-terminal is required for its localization. Association with the PM is mediated by S-acylation of cysteine residues Cys-524 and Cys-527, in a sequence of 45 amino acid residues essential for GSD1 function in rice. Furthermore, the coiled-coil domain in GSD1 is necessary for sufficient interaction with OsACT1. Together, these results reveal that GSD1 attaches to the PM through S-acylation and interacts with OsACT1 through its coiled-coil domain structure to regulate plasmodesmata conductance for photoassimilate transport in rice.

No MeSH data available.


Related in: MedlinePlus

Analysis of the GSD1 domain for binding to OsACT1. (A) GSD1 domains (GSD1N, GSD1C1, or GSD1C2, shown in Figure 2A) are examined for interactions with OsACT1 using bimolecular fluorescence complementation (BiFC) assay. GSD1N: GSD1 N-terminal fragment; GSD1C1, GSD1 C-terminal fragment; GSD1C2: actin-binding domain. Bars = 50 μm. (B) and (C) Interaction of GSD1C1-Myc (B) or GSD1C2-Myc (C) with OsACT1-Flag is detected by Co-immunoprecipitation (Co-IP). GSD1-Myc and OsACT1-FLAG were individually expressed or combinationally co-expressed in tobacco leaves. Total protein extract was immunoprecipitated with anti-Myc antibodies coupled agarose beads or anti-Flag antibodies coupled agarose beads. Proteins from crude lysate and immunoprecipitation were detected with anti-Myc antibodies and anti-Flag antibodies, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4590517&req=5

Figure 5: Analysis of the GSD1 domain for binding to OsACT1. (A) GSD1 domains (GSD1N, GSD1C1, or GSD1C2, shown in Figure 2A) are examined for interactions with OsACT1 using bimolecular fluorescence complementation (BiFC) assay. GSD1N: GSD1 N-terminal fragment; GSD1C1, GSD1 C-terminal fragment; GSD1C2: actin-binding domain. Bars = 50 μm. (B) and (C) Interaction of GSD1C1-Myc (B) or GSD1C2-Myc (C) with OsACT1-Flag is detected by Co-immunoprecipitation (Co-IP). GSD1-Myc and OsACT1-FLAG were individually expressed or combinationally co-expressed in tobacco leaves. Total protein extract was immunoprecipitated with anti-Myc antibodies coupled agarose beads or anti-Flag antibodies coupled agarose beads. Proteins from crude lysate and immunoprecipitation were detected with anti-Myc antibodies and anti-Flag antibodies, respectively.

Mentions: We showed in a previous study that GSD1 affects the grain setting in rice through interacting with OsACT1 to regulate PD conductance. However, the part of the GSD1 structure responsible for interactions with OsACT1 is unknown. To investigate how GSD1 interacts with OsACT1, the full-length GSD1 was truncated to GSD1N (N-terminal part of GSD1), GSD1C1 (C-terminal part of GSD1) and GSD1C2 (the coiled-coil domain and adjacent residues in the C-terminal part), and each truncation was fused to YFP C-terminal sequence (YC), while the YFP N-terminal sequence (YN) moiety was fused with OsACT1. When the GSD1 truncations (YC-GSD1N, YC-GSD1C1 and YC-GSD1C2) were individually co-expressed with YN-OsACT1 in tobacco leaf cells, YC-GSD1C1 and YC-GSD1C2 were shown to interact with YN-OsACT1, however, both free YC and YC-GSD1N were not shown to interact with YN-OsACT1 (Figure 5A, Supplementary Figure S7A). The fluorescent interaction signal was detected predominantly on the PM with an uneven distribution (Figure 5A). These results indicated that the C-terminal region containing the coiled-coil domain in GSD1 acts as an actin-binding domain mediating the interaction with OsACT1. To verify the interaction of the actin-binding domain with OsACT1, the GSD1 was sectioned into GSD1N, GSD1C1, and GSD1C2 fragments used in the subcellular localization and BiFC analyses. Myc-fused GSD1 fragments (GSD1N-Myc, GSD1C1-Myc, and GSD1C2-Myc) and Flag-fused OsACT1 (OsACT1-Flag) were prepared and used for co-immunoprecipitation (Co-IP). As shown in Figure 5B, the anti-Myc antibody-coupled agarose beads were co-precipitated with both OsACT1-Flag and GSD1C1-Myc. Conversely, the anti-Flag antibody-coupled agarose beads were able to co-precipitate both GSD1C1-Myc and the OsACT1-Flag. In contrast, GSD1N-Myc and OsACT1-Flag did not show co-precipitation together (Supplementary Figure S7B). In addition, the anti-Flag antibody-coupled agarose beads were not able to pull down GSD1C1-Myc along with Myosin1-Flag or Myosin2-Flag, which are the cytoskeleton components localized in plasmodesmata (Supplementary Figure S7C). These results demonstrate that the C-terminal region of GSD1 mediated the specific interaction with OsACT1. In addition, the anti-Myc antibody as well as anti-Flag antibody-coupled agarose beads co-precipitated with both GSD1C2-Myc and OsACT1-Flag, further confirming the direct interaction of the GSD1 coiled-coil domain (GSD1C2) with OsACT1 (Figure 5C). Collectively, these results demonstrate that the coiled-coil domain is necessary and sufficient for GSD1 to specifically interact with OsACT1.


Grain setting defect1 (GSD1) function in rice depends on S-acylation and interacts with actin 1 (OsACT1) at its C-terminal.

Gui J, Zheng S, Shen J, Li L - Front Plant Sci (2015)

Analysis of the GSD1 domain for binding to OsACT1. (A) GSD1 domains (GSD1N, GSD1C1, or GSD1C2, shown in Figure 2A) are examined for interactions with OsACT1 using bimolecular fluorescence complementation (BiFC) assay. GSD1N: GSD1 N-terminal fragment; GSD1C1, GSD1 C-terminal fragment; GSD1C2: actin-binding domain. Bars = 50 μm. (B) and (C) Interaction of GSD1C1-Myc (B) or GSD1C2-Myc (C) with OsACT1-Flag is detected by Co-immunoprecipitation (Co-IP). GSD1-Myc and OsACT1-FLAG were individually expressed or combinationally co-expressed in tobacco leaves. Total protein extract was immunoprecipitated with anti-Myc antibodies coupled agarose beads or anti-Flag antibodies coupled agarose beads. Proteins from crude lysate and immunoprecipitation were detected with anti-Myc antibodies and anti-Flag antibodies, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4590517&req=5

Figure 5: Analysis of the GSD1 domain for binding to OsACT1. (A) GSD1 domains (GSD1N, GSD1C1, or GSD1C2, shown in Figure 2A) are examined for interactions with OsACT1 using bimolecular fluorescence complementation (BiFC) assay. GSD1N: GSD1 N-terminal fragment; GSD1C1, GSD1 C-terminal fragment; GSD1C2: actin-binding domain. Bars = 50 μm. (B) and (C) Interaction of GSD1C1-Myc (B) or GSD1C2-Myc (C) with OsACT1-Flag is detected by Co-immunoprecipitation (Co-IP). GSD1-Myc and OsACT1-FLAG were individually expressed or combinationally co-expressed in tobacco leaves. Total protein extract was immunoprecipitated with anti-Myc antibodies coupled agarose beads or anti-Flag antibodies coupled agarose beads. Proteins from crude lysate and immunoprecipitation were detected with anti-Myc antibodies and anti-Flag antibodies, respectively.
Mentions: We showed in a previous study that GSD1 affects the grain setting in rice through interacting with OsACT1 to regulate PD conductance. However, the part of the GSD1 structure responsible for interactions with OsACT1 is unknown. To investigate how GSD1 interacts with OsACT1, the full-length GSD1 was truncated to GSD1N (N-terminal part of GSD1), GSD1C1 (C-terminal part of GSD1) and GSD1C2 (the coiled-coil domain and adjacent residues in the C-terminal part), and each truncation was fused to YFP C-terminal sequence (YC), while the YFP N-terminal sequence (YN) moiety was fused with OsACT1. When the GSD1 truncations (YC-GSD1N, YC-GSD1C1 and YC-GSD1C2) were individually co-expressed with YN-OsACT1 in tobacco leaf cells, YC-GSD1C1 and YC-GSD1C2 were shown to interact with YN-OsACT1, however, both free YC and YC-GSD1N were not shown to interact with YN-OsACT1 (Figure 5A, Supplementary Figure S7A). The fluorescent interaction signal was detected predominantly on the PM with an uneven distribution (Figure 5A). These results indicated that the C-terminal region containing the coiled-coil domain in GSD1 acts as an actin-binding domain mediating the interaction with OsACT1. To verify the interaction of the actin-binding domain with OsACT1, the GSD1 was sectioned into GSD1N, GSD1C1, and GSD1C2 fragments used in the subcellular localization and BiFC analyses. Myc-fused GSD1 fragments (GSD1N-Myc, GSD1C1-Myc, and GSD1C2-Myc) and Flag-fused OsACT1 (OsACT1-Flag) were prepared and used for co-immunoprecipitation (Co-IP). As shown in Figure 5B, the anti-Myc antibody-coupled agarose beads were co-precipitated with both OsACT1-Flag and GSD1C1-Myc. Conversely, the anti-Flag antibody-coupled agarose beads were able to co-precipitate both GSD1C1-Myc and the OsACT1-Flag. In contrast, GSD1N-Myc and OsACT1-Flag did not show co-precipitation together (Supplementary Figure S7B). In addition, the anti-Flag antibody-coupled agarose beads were not able to pull down GSD1C1-Myc along with Myosin1-Flag or Myosin2-Flag, which are the cytoskeleton components localized in plasmodesmata (Supplementary Figure S7C). These results demonstrate that the C-terminal region of GSD1 mediated the specific interaction with OsACT1. In addition, the anti-Myc antibody as well as anti-Flag antibody-coupled agarose beads co-precipitated with both GSD1C2-Myc and OsACT1-Flag, further confirming the direct interaction of the GSD1 coiled-coil domain (GSD1C2) with OsACT1 (Figure 5C). Collectively, these results demonstrate that the coiled-coil domain is necessary and sufficient for GSD1 to specifically interact with OsACT1.

Bottom Line: Association with the PM is mediated by S-acylation of cysteine residues Cys-524 and Cys-527, in a sequence of 45 amino acid residues essential for GSD1 function in rice.Furthermore, the coiled-coil domain in GSD1 is necessary for sufficient interaction with OsACT1.Together, these results reveal that GSD1 attaches to the PM through S-acylation and interacts with OsACT1 through its coiled-coil domain structure to regulate plasmodesmata conductance for photoassimilate transport in rice.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Shanghai, China.

ABSTRACT
Grain setting defect1 (GSD1), a plant-specific remorin protein specifically localized at the plasma membrane (PM) and plasmodesmata of phloem companion cells, affects grain setting in rice through regulating the transport of photoassimilates. Here, we show new evidence demonstrating that GSD1 is localized at the cytoplasmic face of the PM and a stretch of 45 amino acid residues at its C-terminal is required for its localization. Association with the PM is mediated by S-acylation of cysteine residues Cys-524 and Cys-527, in a sequence of 45 amino acid residues essential for GSD1 function in rice. Furthermore, the coiled-coil domain in GSD1 is necessary for sufficient interaction with OsACT1. Together, these results reveal that GSD1 attaches to the PM through S-acylation and interacts with OsACT1 through its coiled-coil domain structure to regulate plasmodesmata conductance for photoassimilate transport in rice.

No MeSH data available.


Related in: MedlinePlus