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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: 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.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.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

A stretch of 45 amino acid residues at the C-terminal is sufficient for grain setting defect 1 (GSD1) association with PM. (A) Schematic representation of full-length and truncated GSD1 fragments. CCD, coiled-coil domain. (B) Full-length and truncated GSD1 fragments were fused with GFP and expressed in tobacco leaves. Images show that GSD1C1 and GSD1C3 are localized specifically on PM. Bars = 50 μm. (C) Western blot detection of GFP-GSD1C3, GFP-GSD1C4, and GFP-GSD1C5 in soluble and membrane fractions of the transformed tobacco leaves. T, total; S, soluble; MF, membrane fractions.
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Figure 1: A stretch of 45 amino acid residues at the C-terminal is sufficient for grain setting defect 1 (GSD1) association with PM. (A) Schematic representation of full-length and truncated GSD1 fragments. CCD, coiled-coil domain. (B) Full-length and truncated GSD1 fragments were fused with GFP and expressed in tobacco leaves. Images show that GSD1C1 and GSD1C3 are localized specifically on PM. Bars = 50 μm. (C) Western blot detection of GFP-GSD1C3, GFP-GSD1C4, and GFP-GSD1C5 in soluble and membrane fractions of the transformed tobacco leaves. T, total; S, soluble; MF, membrane fractions.

Mentions: The C-terminal region contains a predicted coiled-coil domain and a variable C-terminus tail. The N-terminal region is predicted to contain a putative GSK3 phosphorylation recognition site, a predicted Tyr-based sorting signal, a putative MAPK interacting motif, which may be involved in specific interactions in MAPK cascades or could be responsible for interactions within the protein complex (Supplementary Figure S2A). To identify which part of GSD1 is responsible for its PM localization, we generated a series of truncated GSD1 proteins (N-terminal part of GSD1: GSD1N, various C-terminal sections of GSD1: GSD1C1, GSD1C2, GSD1C3, GSD1C4, GSD1C5, and GSD1C6, Figure 1A), which were fused with a GFP at the N terminus and used for the examination of their cellular localization. The N-terminal part of GSD1, GSD1N, was localized in the cytoplasm, while GSD1C1, the part deleted with N-terminal, was associated with the PM, at the same location as the full-length GSD1 protein (Figure 1B). This result indicates that the GSD1 PM anchoring sequence is in its C-terminal. To map the exact PM anchoring sequence, further deletion analysis of a series of GSD1 truncated proteins was carried out. When GSD1C1 was cut into GSD1C2 and GSD1C3, GSD1C2 was localized in the cytoplasm, whereas GSD1C3 was exclusively associated with the PM (Figure 1B). As GSD1C3 was further divided into GSD1C4 and GSD1C5, GSD1C4 was present in cytoplasm, while GSD1C5 was predominantly localized on PM but also displayed weak signals in cytoplasm (Figure 1B). However, GSD1C6, with a stretch of 12 amino acid residues removed from the GSD1C1 end, was localized in the cytoplasm (Figure 1B). Taken together, these results demonstrate that a stretch of 12 amino acid residues is necessary but potentially not sufficient for GSD1 to be specifically associated with the PM. In addition to the 12 amino acid residues, other adjacent sequence may be needed to secure GSD1 on PM. Therefore, the distribution of GSD1C3, GSD1C4, and GSD1C5 in the cytoplasm and membrane was examined using plasmolysis and immunoblotting analysis. After plasmolysis, GSD1C3 showed a clear PM localization, while GSD1C4 displayed a typical cytoplasmic distribution (Supplementary Figure S3). GSD1C5 was predominantly localized on the PM, but also exist in cytoplasm (Supplementary Figure S3). Protein immunoblotting analysis using anti-GFP monoclonal antibodies indicated that GFP-GSD1C3 was predominantly localized in the membrane fraction, whereas GFP-GSD1C4 was localized in the soluble fraction (Figure 1C). GFP-GSD1C5 was predominantly present in the membrane fraction, but also present in small amounts in the soluble fraction (Figure 1C). Collectively, GSD1 truncation, plasmolysis and immunoblotting analyses revealed that GSD1C3, essentially a stretch of 45 amino acid residues, which is not conserved among remorins, is sufficient for GSD1 to anchor on the PM.


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)

A stretch of 45 amino acid residues at the C-terminal is sufficient for grain setting defect 1 (GSD1) association with PM. (A) Schematic representation of full-length and truncated GSD1 fragments. CCD, coiled-coil domain. (B) Full-length and truncated GSD1 fragments were fused with GFP and expressed in tobacco leaves. Images show that GSD1C1 and GSD1C3 are localized specifically on PM. Bars = 50 μm. (C) Western blot detection of GFP-GSD1C3, GFP-GSD1C4, and GFP-GSD1C5 in soluble and membrane fractions of the transformed tobacco leaves. T, total; S, soluble; MF, membrane fractions.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: A stretch of 45 amino acid residues at the C-terminal is sufficient for grain setting defect 1 (GSD1) association with PM. (A) Schematic representation of full-length and truncated GSD1 fragments. CCD, coiled-coil domain. (B) Full-length and truncated GSD1 fragments were fused with GFP and expressed in tobacco leaves. Images show that GSD1C1 and GSD1C3 are localized specifically on PM. Bars = 50 μm. (C) Western blot detection of GFP-GSD1C3, GFP-GSD1C4, and GFP-GSD1C5 in soluble and membrane fractions of the transformed tobacco leaves. T, total; S, soluble; MF, membrane fractions.
Mentions: The C-terminal region contains a predicted coiled-coil domain and a variable C-terminus tail. The N-terminal region is predicted to contain a putative GSK3 phosphorylation recognition site, a predicted Tyr-based sorting signal, a putative MAPK interacting motif, which may be involved in specific interactions in MAPK cascades or could be responsible for interactions within the protein complex (Supplementary Figure S2A). To identify which part of GSD1 is responsible for its PM localization, we generated a series of truncated GSD1 proteins (N-terminal part of GSD1: GSD1N, various C-terminal sections of GSD1: GSD1C1, GSD1C2, GSD1C3, GSD1C4, GSD1C5, and GSD1C6, Figure 1A), which were fused with a GFP at the N terminus and used for the examination of their cellular localization. The N-terminal part of GSD1, GSD1N, was localized in the cytoplasm, while GSD1C1, the part deleted with N-terminal, was associated with the PM, at the same location as the full-length GSD1 protein (Figure 1B). This result indicates that the GSD1 PM anchoring sequence is in its C-terminal. To map the exact PM anchoring sequence, further deletion analysis of a series of GSD1 truncated proteins was carried out. When GSD1C1 was cut into GSD1C2 and GSD1C3, GSD1C2 was localized in the cytoplasm, whereas GSD1C3 was exclusively associated with the PM (Figure 1B). As GSD1C3 was further divided into GSD1C4 and GSD1C5, GSD1C4 was present in cytoplasm, while GSD1C5 was predominantly localized on PM but also displayed weak signals in cytoplasm (Figure 1B). However, GSD1C6, with a stretch of 12 amino acid residues removed from the GSD1C1 end, was localized in the cytoplasm (Figure 1B). Taken together, these results demonstrate that a stretch of 12 amino acid residues is necessary but potentially not sufficient for GSD1 to be specifically associated with the PM. In addition to the 12 amino acid residues, other adjacent sequence may be needed to secure GSD1 on PM. Therefore, the distribution of GSD1C3, GSD1C4, and GSD1C5 in the cytoplasm and membrane was examined using plasmolysis and immunoblotting analysis. After plasmolysis, GSD1C3 showed a clear PM localization, while GSD1C4 displayed a typical cytoplasmic distribution (Supplementary Figure S3). GSD1C5 was predominantly localized on the PM, but also exist in cytoplasm (Supplementary Figure S3). Protein immunoblotting analysis using anti-GFP monoclonal antibodies indicated that GFP-GSD1C3 was predominantly localized in the membrane fraction, whereas GFP-GSD1C4 was localized in the soluble fraction (Figure 1C). GFP-GSD1C5 was predominantly present in the membrane fraction, but also present in small amounts in the soluble fraction (Figure 1C). Collectively, GSD1 truncation, plasmolysis and immunoblotting analyses revealed that GSD1C3, essentially a stretch of 45 amino acid residues, which is not conserved among remorins, is sufficient for GSD1 to anchor on the PM.

Bottom Line: 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.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.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