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A green fluorescent protein fused to rice prolamin forms protein body-like structures in transgenic rice.

Saito Y, Kishida K, Takata K, Takahashi H, Shimada T, Tanaka K, Morita S, Satoh S, Masumura T - J. Exp. Bot. (2009)

Bottom Line: The ER chaperone BiP was detected in the structures in the leaves and roots.The results show that the aggregation of prolamin-GFP fusion proteins does not depend on the tissues, suggesting that the prolamin-GFP fusion proteins accumulate in the ER by forming into aggregates.The findings bear out the importance of the assembly of prolamin molecules and the interaction of prolamin with BiP in the formation of ER-derived PBs.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan.

ABSTRACT
Prolamins, a group of rice (Oryza sativa) seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and deposited in ER-derived type I protein bodies (PB-Is) in rice endosperm cells. The accumulation mechanism of prolamins, which do not possess the well-known ER retention signal, remains unclear. In order to elucidate whether the accumulation of prolamin in the ER requires seed-specific factors, the subcellular localization of the constitutively expressed green fluorescent protein fused to prolamin (prolamin-GFP) was examined in seeds, leaves, and roots of transgenic rice plants. The prolamin-GFP fusion proteins accumulated not only in the seeds but also in the leaves and roots. Microscopic observation of GFP fluorescence and immunocytochemical analysis revealed that prolamin-GFP fusion proteins specifically accumulated in PB-Is in the endosperm, whereas they were deposited in the electron-dense structures in the leaves and roots. The ER chaperone BiP was detected in the structures in the leaves and roots. The results show that the aggregation of prolamin-GFP fusion proteins does not depend on the tissues, suggesting that the prolamin-GFP fusion proteins accumulate in the ER by forming into aggregates. The findings bear out the importance of the assembly of prolamin molecules and the interaction of prolamin with BiP in the formation of ER-derived PBs.

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Influence of 2-ME on prolamin–GFP fusion protein solubilization. In the experiment shown in A, proteins from seeds of WT (lanes 1 and 4), 35S:GFP (lanes 2 and 5), and 35S:Pro-GFP (lanes 3 and 6) plants were extracted with 5% (v/v) 2-ME (lanes 1–3) or without 2-ME (lanes 4–6). In B, homogenates from leaves of WT (lanes 1 and 4), 35S:GFP (lanes 2 and 5), and 35S:Pro-GFP (lanes 3 and 6) plants were subjected to centrifugation to obtain the 15 000 g pellet (P15) and the 15 000 g supernatant (S15). Proteins in each fraction were extracted with 5% (v/v) 2-ME (lanes 1–3) or without 2-ME (lanes 4–6). The proteins were separated by SDS–PAGE and immunoblotted with anti-GFP antibodies. The upper bands (open arrowheads) and lower bands (filled arrowheads) correspond to prolamin–GFP and GFP, respectively.
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fig9: Influence of 2-ME on prolamin–GFP fusion protein solubilization. In the experiment shown in A, proteins from seeds of WT (lanes 1 and 4), 35S:GFP (lanes 2 and 5), and 35S:Pro-GFP (lanes 3 and 6) plants were extracted with 5% (v/v) 2-ME (lanes 1–3) or without 2-ME (lanes 4–6). In B, homogenates from leaves of WT (lanes 1 and 4), 35S:GFP (lanes 2 and 5), and 35S:Pro-GFP (lanes 3 and 6) plants were subjected to centrifugation to obtain the 15 000 g pellet (P15) and the 15 000 g supernatant (S15). Proteins in each fraction were extracted with 5% (v/v) 2-ME (lanes 1–3) or without 2-ME (lanes 4–6). The proteins were separated by SDS–PAGE and immunoblotted with anti-GFP antibodies. The upper bands (open arrowheads) and lower bands (filled arrowheads) correspond to prolamin–GFP and GFP, respectively.

Mentions: The rice 13 kDa prolamin encoded by λRM1 contains four cysteine residues. Because little is known about which cysteine residues in rice prolamins engage in intermolecular and/or intramolecular disulphide bonds, an investigation was carried out to determine whether cysteine residues of prolamin–GFP fusion proteins could form intermolecular disulphide bonds. In the seeds of 35S:Pro-GFP plants, the addition of 4% 2-ME to the extraction buffer reduced the number of disulphide bonds, leading to prolamin–GFP solubilization (Fig. 9A, lane 3, open arrowhead). When proteins were extracted from seeds of 35S:Pro-GFP plants in the absence of 2-ME, prolamin–GFP was not solubilized (Fig. 9A, lane 6). The presence or absence of 2-ME did not affect the GFP solubilization in protein extracts from the seeds of 35S:GFP plants (Fig. 9A, lanes 2 and 5, closed arrowhead). These results suggest that the cysteine residues of prolamin–GFP formed intermolecular disulphide bonds with prolamin–GFP in the endosperm cells. To investigate whether the cysteine residues of prolamin–GFP expressed in the leaves could form intermolecular disulphide bonds with prolamin–GFP molecules, the influence of 2-ME on the solubility of prolamin–GFP fusion proteins in the leaves was examined. The homogenates from leaves were separated into two subcellular fractions: a 15 000 g pellet (P15; the PB fractions) and a 15 000 g supernatant (S15). When the proteins in the P15 fractions from 35S:Pro-GFP plants were extracted in the presence or absence of 2-ME, prolamin–GFP was solubilized under reducing conditions, whereas the prolamin–GFP was only partially solubilized under non-reducing conditions (Fig. 9B, lanes 3 and 6, open arrowhead). In contrast, GFP was not affected by the absence of 2-ME in the S15 fractions from 35S:GFP plants (Fig. 9B, lanes 2 and 4, closed arrowhead). These results indicate that the prolamin–GFP fusion proteins in the PB-like structures form the intermolecular disulphide bonds.


A green fluorescent protein fused to rice prolamin forms protein body-like structures in transgenic rice.

Saito Y, Kishida K, Takata K, Takahashi H, Shimada T, Tanaka K, Morita S, Satoh S, Masumura T - J. Exp. Bot. (2009)

Influence of 2-ME on prolamin–GFP fusion protein solubilization. In the experiment shown in A, proteins from seeds of WT (lanes 1 and 4), 35S:GFP (lanes 2 and 5), and 35S:Pro-GFP (lanes 3 and 6) plants were extracted with 5% (v/v) 2-ME (lanes 1–3) or without 2-ME (lanes 4–6). In B, homogenates from leaves of WT (lanes 1 and 4), 35S:GFP (lanes 2 and 5), and 35S:Pro-GFP (lanes 3 and 6) plants were subjected to centrifugation to obtain the 15 000 g pellet (P15) and the 15 000 g supernatant (S15). Proteins in each fraction were extracted with 5% (v/v) 2-ME (lanes 1–3) or without 2-ME (lanes 4–6). The proteins were separated by SDS–PAGE and immunoblotted with anti-GFP antibodies. The upper bands (open arrowheads) and lower bands (filled arrowheads) correspond to prolamin–GFP and GFP, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2651459&req=5

fig9: Influence of 2-ME on prolamin–GFP fusion protein solubilization. In the experiment shown in A, proteins from seeds of WT (lanes 1 and 4), 35S:GFP (lanes 2 and 5), and 35S:Pro-GFP (lanes 3 and 6) plants were extracted with 5% (v/v) 2-ME (lanes 1–3) or without 2-ME (lanes 4–6). In B, homogenates from leaves of WT (lanes 1 and 4), 35S:GFP (lanes 2 and 5), and 35S:Pro-GFP (lanes 3 and 6) plants were subjected to centrifugation to obtain the 15 000 g pellet (P15) and the 15 000 g supernatant (S15). Proteins in each fraction were extracted with 5% (v/v) 2-ME (lanes 1–3) or without 2-ME (lanes 4–6). The proteins were separated by SDS–PAGE and immunoblotted with anti-GFP antibodies. The upper bands (open arrowheads) and lower bands (filled arrowheads) correspond to prolamin–GFP and GFP, respectively.
Mentions: The rice 13 kDa prolamin encoded by λRM1 contains four cysteine residues. Because little is known about which cysteine residues in rice prolamins engage in intermolecular and/or intramolecular disulphide bonds, an investigation was carried out to determine whether cysteine residues of prolamin–GFP fusion proteins could form intermolecular disulphide bonds. In the seeds of 35S:Pro-GFP plants, the addition of 4% 2-ME to the extraction buffer reduced the number of disulphide bonds, leading to prolamin–GFP solubilization (Fig. 9A, lane 3, open arrowhead). When proteins were extracted from seeds of 35S:Pro-GFP plants in the absence of 2-ME, prolamin–GFP was not solubilized (Fig. 9A, lane 6). The presence or absence of 2-ME did not affect the GFP solubilization in protein extracts from the seeds of 35S:GFP plants (Fig. 9A, lanes 2 and 5, closed arrowhead). These results suggest that the cysteine residues of prolamin–GFP formed intermolecular disulphide bonds with prolamin–GFP in the endosperm cells. To investigate whether the cysteine residues of prolamin–GFP expressed in the leaves could form intermolecular disulphide bonds with prolamin–GFP molecules, the influence of 2-ME on the solubility of prolamin–GFP fusion proteins in the leaves was examined. The homogenates from leaves were separated into two subcellular fractions: a 15 000 g pellet (P15; the PB fractions) and a 15 000 g supernatant (S15). When the proteins in the P15 fractions from 35S:Pro-GFP plants were extracted in the presence or absence of 2-ME, prolamin–GFP was solubilized under reducing conditions, whereas the prolamin–GFP was only partially solubilized under non-reducing conditions (Fig. 9B, lanes 3 and 6, open arrowhead). In contrast, GFP was not affected by the absence of 2-ME in the S15 fractions from 35S:GFP plants (Fig. 9B, lanes 2 and 4, closed arrowhead). These results indicate that the prolamin–GFP fusion proteins in the PB-like structures form the intermolecular disulphide bonds.

Bottom Line: The ER chaperone BiP was detected in the structures in the leaves and roots.The results show that the aggregation of prolamin-GFP fusion proteins does not depend on the tissues, suggesting that the prolamin-GFP fusion proteins accumulate in the ER by forming into aggregates.The findings bear out the importance of the assembly of prolamin molecules and the interaction of prolamin with BiP in the formation of ER-derived PBs.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan.

ABSTRACT
Prolamins, a group of rice (Oryza sativa) seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and deposited in ER-derived type I protein bodies (PB-Is) in rice endosperm cells. The accumulation mechanism of prolamins, which do not possess the well-known ER retention signal, remains unclear. In order to elucidate whether the accumulation of prolamin in the ER requires seed-specific factors, the subcellular localization of the constitutively expressed green fluorescent protein fused to prolamin (prolamin-GFP) was examined in seeds, leaves, and roots of transgenic rice plants. The prolamin-GFP fusion proteins accumulated not only in the seeds but also in the leaves and roots. Microscopic observation of GFP fluorescence and immunocytochemical analysis revealed that prolamin-GFP fusion proteins specifically accumulated in PB-Is in the endosperm, whereas they were deposited in the electron-dense structures in the leaves and roots. The ER chaperone BiP was detected in the structures in the leaves and roots. The results show that the aggregation of prolamin-GFP fusion proteins does not depend on the tissues, suggesting that the prolamin-GFP fusion proteins accumulate in the ER by forming into aggregates. The findings bear out the importance of the assembly of prolamin molecules and the interaction of prolamin with BiP in the formation of ER-derived PBs.

Show MeSH
Related in: MedlinePlus