The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Takehiro Masumura - One of the best experts on this subject based on the ideXlab platform.
-
The localization of rice prolamin species in protein body type I is determined by the temporal control of gene expression of the respective prolamin promoters.
Plant biotechnology (Tokyo Japan), 2018Co-Authors: Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Takehiro MasumuraAbstract:Rice prolamin species form a layered structure in the protein body type I (PB-I) storage organelle. Rice Prolamins are classified as 10 kDa, 13a-1, 13a-2, 13b-1, 13b-2 and 16 kDa prolamin. Prolamin species form layer structure in PB-I in order of 10 kDa core, 13b-1 layer, 13a (13a-1 and 13a-2) and 16 kDa middle layer and 13b-2 outer-most layer. In a previous study, we showed that the fusion proteins in 13b-2 prolamin-GFP, 13a-1 prolamin-GFP and 10 kDa prolamin-GFP were localized in the same layer of PB-I as the native prolamin, when they were expressed by their respective native prolamin promoters. Our preliminary study suggested that the temporal control of the native prolamin promoters was responsible for the localization of the respective Prolamins. The aim of this study was to determine whether the use of a prolamin promoter other than the native prolamin promoter would change the localization of prolamin-GFP fusion proteins. For this purpose, we generated transgenic lines expressing 13b-2 prolamin-GFP and 13a-1 prolamin-GFP fusion proteins driven by each prolamin promoter other than the native prolamin promoter. As a result, the localization of the fusion protein in PB-I was changed. Based on our results, foreign protein localization in PB-I can be achieved by the temporal control of the different prolamin promoters.
-
Accumulation of foreign polypeptides to rice seed protein body type I using prolamin portion sequences.
Plant cell reports, 2016Co-Authors: Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Takehiro MasumuraAbstract:Rice Prolamins are accumulated in endoplasmic reticulum (ER)-derived proteins bodies, although conserved sequences retained in ER are not confirmed. We investigated portion sequences of Prolamins that must accumulate in PB-Is. Rice seed Prolamins are accumulated in endoplasmic reticulum (ER)-derived protein body type I (PB-I), but ER retention sequences in rice prolamin polypeptides have not been confirmed. Here we investigated the lengths of the prolamin portion sequences required for accumulation in PB-Is. Of the rice Prolamins, we compared 13a and 13b Prolamins because the amino acid sequences of these Prolamins are quite similar except for the presence or absence of Cys-residues. We also generated and analyzed transgenic rice expressing several prolamin portion sequence-GFP fusion proteins. We observed that in 13a prolamin, when the portion sequences were extended more than the 68th amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. In 13b prolamin, when the portion sequences were extended by more than the 82nd amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. When those fusion proteins were extracted under non-reduced or reduced conditions, the 13a prolamin portion sequence-GFP fusion proteins in PB-Is were soluble under only the reduced condition. In contrast, 13b prolamin portion sequence-GFP fusion proteins were soluble under both non-reduced and reduced conditions. These results suggest that the accumulation of 13a prolamin in PB-Is is associated with the formation of disulfide bonds and/or hydrophobicity in 13a prolamin polypeptide, whereas the accumulation of 13b prolamin in PB-Is was less involved in the formation of disulfide bonds.
-
Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system.
Bioscience biotechnology and biochemistry, 2014Co-Authors: Takehiro Masumura, Takanari Shigemitsu, Shigeto Morita, Shigeru SatohAbstract:Cereal Prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat Prolamins, rice Prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.
-
Accumulation of rice prolamin–GFP fusion proteins induces ER-derived protein bodies in transgenic rice calli
Plant cell reports, 2012Co-Authors: Takanari Shigemitsu, Takehiro Masumura, Shigeto Morita, Shigeru SatohAbstract:We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Rice Prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice Prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat Prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin–GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin–GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.
-
A green fluorescent protein fused to rice prolamin forms protein body-like structures in transgenic rice
Journal of experimental botany, 2009Co-Authors: Yuhi Saito, Kunisuke Tanaka, Shigeto Morita, Koichi Kishida, Shigeru Satoh, Kenji Takata, Hideyuki Takahashi, Takeaki Shimada, Takehiro MasumuraAbstract: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.
Shigeru Satoh - One of the best experts on this subject based on the ideXlab platform.
-
Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system.
Bioscience biotechnology and biochemistry, 2014Co-Authors: Takehiro Masumura, Takanari Shigemitsu, Shigeto Morita, Shigeru SatohAbstract:Cereal Prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat Prolamins, rice Prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.
-
Accumulation of rice prolamin–GFP fusion proteins induces ER-derived protein bodies in transgenic rice calli
Plant cell reports, 2012Co-Authors: Takanari Shigemitsu, Takehiro Masumura, Shigeto Morita, Shigeru SatohAbstract:We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Rice Prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice Prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat Prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin–GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin–GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.
-
Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes
The Plant journal : for cell and molecular biology, 2012Co-Authors: Yuhi Saito, Kunisuke Tanaka, Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Ryuichi Yamasaki, Futami Goto, Koichi Kishida, Masaharu Kuroda, Shigeru SatohAbstract:Rice Prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice Prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa Prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b Prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.
-
A green fluorescent protein fused to rice prolamin forms protein body-like structures in transgenic rice
Journal of experimental botany, 2009Co-Authors: Yuhi Saito, Kunisuke Tanaka, Shigeto Morita, Koichi Kishida, Shigeru Satoh, Kenji Takata, Hideyuki Takahashi, Takeaki Shimada, Takehiro MasumuraAbstract: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.
Shigeto Morita - One of the best experts on this subject based on the ideXlab platform.
-
The localization of rice prolamin species in protein body type I is determined by the temporal control of gene expression of the respective prolamin promoters.
Plant biotechnology (Tokyo Japan), 2018Co-Authors: Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Takehiro MasumuraAbstract:Rice prolamin species form a layered structure in the protein body type I (PB-I) storage organelle. Rice Prolamins are classified as 10 kDa, 13a-1, 13a-2, 13b-1, 13b-2 and 16 kDa prolamin. Prolamin species form layer structure in PB-I in order of 10 kDa core, 13b-1 layer, 13a (13a-1 and 13a-2) and 16 kDa middle layer and 13b-2 outer-most layer. In a previous study, we showed that the fusion proteins in 13b-2 prolamin-GFP, 13a-1 prolamin-GFP and 10 kDa prolamin-GFP were localized in the same layer of PB-I as the native prolamin, when they were expressed by their respective native prolamin promoters. Our preliminary study suggested that the temporal control of the native prolamin promoters was responsible for the localization of the respective Prolamins. The aim of this study was to determine whether the use of a prolamin promoter other than the native prolamin promoter would change the localization of prolamin-GFP fusion proteins. For this purpose, we generated transgenic lines expressing 13b-2 prolamin-GFP and 13a-1 prolamin-GFP fusion proteins driven by each prolamin promoter other than the native prolamin promoter. As a result, the localization of the fusion protein in PB-I was changed. Based on our results, foreign protein localization in PB-I can be achieved by the temporal control of the different prolamin promoters.
-
Accumulation of foreign polypeptides to rice seed protein body type I using prolamin portion sequences.
Plant cell reports, 2016Co-Authors: Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Takehiro MasumuraAbstract:Rice Prolamins are accumulated in endoplasmic reticulum (ER)-derived proteins bodies, although conserved sequences retained in ER are not confirmed. We investigated portion sequences of Prolamins that must accumulate in PB-Is. Rice seed Prolamins are accumulated in endoplasmic reticulum (ER)-derived protein body type I (PB-I), but ER retention sequences in rice prolamin polypeptides have not been confirmed. Here we investigated the lengths of the prolamin portion sequences required for accumulation in PB-Is. Of the rice Prolamins, we compared 13a and 13b Prolamins because the amino acid sequences of these Prolamins are quite similar except for the presence or absence of Cys-residues. We also generated and analyzed transgenic rice expressing several prolamin portion sequence-GFP fusion proteins. We observed that in 13a prolamin, when the portion sequences were extended more than the 68th amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. In 13b prolamin, when the portion sequences were extended by more than the 82nd amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. When those fusion proteins were extracted under non-reduced or reduced conditions, the 13a prolamin portion sequence-GFP fusion proteins in PB-Is were soluble under only the reduced condition. In contrast, 13b prolamin portion sequence-GFP fusion proteins were soluble under both non-reduced and reduced conditions. These results suggest that the accumulation of 13a prolamin in PB-Is is associated with the formation of disulfide bonds and/or hydrophobicity in 13a prolamin polypeptide, whereas the accumulation of 13b prolamin in PB-Is was less involved in the formation of disulfide bonds.
-
Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system.
Bioscience biotechnology and biochemistry, 2014Co-Authors: Takehiro Masumura, Takanari Shigemitsu, Shigeto Morita, Shigeru SatohAbstract:Cereal Prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat Prolamins, rice Prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.
-
Accumulation of rice prolamin–GFP fusion proteins induces ER-derived protein bodies in transgenic rice calli
Plant cell reports, 2012Co-Authors: Takanari Shigemitsu, Takehiro Masumura, Shigeto Morita, Shigeru SatohAbstract:We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Rice Prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice Prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat Prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin–GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin–GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.
-
Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes
The Plant journal : for cell and molecular biology, 2012Co-Authors: Yuhi Saito, Kunisuke Tanaka, Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Ryuichi Yamasaki, Futami Goto, Koichi Kishida, Masaharu Kuroda, Shigeru SatohAbstract:Rice Prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice Prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa Prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b Prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.
Kenjiro Ozawa - One of the best experts on this subject based on the ideXlab platform.
-
Compensatory rebalancing of rice Prolamins by production of recombinant prolamin/bioactive peptide fusion proteins within ER-derived protein bodies
Plant Cell Reports, 2018Co-Authors: Fumio Takaiwa, Yoshio Wakasa, Lijun Yang, Kenjiro OzawaAbstract:Key messageBioactive peptide was produced by fusion to rice Prolamins in transgenic rice seeds. Their accumulation levels were affected by their deposition sites and by compensatory rebalancing between Prolamins within PB-Is .AbstractPeptide immunotherapy using analogue peptide ligands (APLs) is one of promising treatments against autoimmune diseases. Use of seed storage protein as a fusion carrier is reasonable strategy for production of such small size bioactive peptides. In this study, to examine the efficacy of various rice Prolamins deposited in ER-derived protein bodies (PB-Is), the APL12 from the Glucose-6-phosphate isomerase (GPI325-339) was expressed by fusion to four types of representative Prolamins under the control of the individual native promoters. When the 14 and 16 kDa Cys-rich Prolamins, which were localized in middle layer of PB-Is, were used for production of the APL12, they highly accumulated in transgenic rice seeds (~ 200 µg/grain). By contrast, fusion to the 10 and 13 kDa Prolamins, which were localized in the core and outermost layer of PB-Is, resulted in lower levels of accumulation (~ 40 µg/grain). These results suggest that accumulation levels were highly affected by their deposition sites. Next, when different prolamin/APL12 fusion proteins were co-expressed to increase accumulation levels, they could not be increased so much as their expected additive levels. High accumulation of one type prolamin/APL12 led to reduction of other type(s) prolamin/APL12 to maintain the limited amounts of Prolamins that can be deposited in PB-Is. Moreover, suppression of endogenous seed proteins by RNA interference also did not significantly enhance the accumulation levels of prolamin/APL12. These findings suggest that there may be compensatory rebalancing mechanism that controls the accumulation levels of Prolamins deposited within PB-Is.
-
compensatory rebalancing of rice Prolamins by production of recombinant prolamin bioactive peptide fusion proteins within er derived protein bodies
Plant Cell Reports, 2018Co-Authors: Fumio Takaiwa, Lijun Yang, Yuhya Wakasa, Kenjiro OzawaAbstract:Key message Bioactive peptide was produced by fusion to rice Prolamins in transgenic rice seeds. Their accumulation levels were affected by their deposition sites and by compensatory rebalancing between Prolamins within PB-Is.
-
Compensatory rebalancing of rice Prolamins by production of recombinant prolamin/bioactive peptide fusion proteins within ER-derived protein bodies.
Plant cell reports, 2017Co-Authors: Fumio Takaiwa, Lijun Yang, Yuhya Wakasa, Kenjiro OzawaAbstract:Key message Bioactive peptide was produced by fusion to rice Prolamins in transgenic rice seeds. Their accumulation levels were affected by their deposition sites and by compensatory rebalancing between Prolamins within PB-Is.
Takanari Shigemitsu - One of the best experts on this subject based on the ideXlab platform.
-
The localization of rice prolamin species in protein body type I is determined by the temporal control of gene expression of the respective prolamin promoters.
Plant biotechnology (Tokyo Japan), 2018Co-Authors: Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Takehiro MasumuraAbstract:Rice prolamin species form a layered structure in the protein body type I (PB-I) storage organelle. Rice Prolamins are classified as 10 kDa, 13a-1, 13a-2, 13b-1, 13b-2 and 16 kDa prolamin. Prolamin species form layer structure in PB-I in order of 10 kDa core, 13b-1 layer, 13a (13a-1 and 13a-2) and 16 kDa middle layer and 13b-2 outer-most layer. In a previous study, we showed that the fusion proteins in 13b-2 prolamin-GFP, 13a-1 prolamin-GFP and 10 kDa prolamin-GFP were localized in the same layer of PB-I as the native prolamin, when they were expressed by their respective native prolamin promoters. Our preliminary study suggested that the temporal control of the native prolamin promoters was responsible for the localization of the respective Prolamins. The aim of this study was to determine whether the use of a prolamin promoter other than the native prolamin promoter would change the localization of prolamin-GFP fusion proteins. For this purpose, we generated transgenic lines expressing 13b-2 prolamin-GFP and 13a-1 prolamin-GFP fusion proteins driven by each prolamin promoter other than the native prolamin promoter. As a result, the localization of the fusion protein in PB-I was changed. Based on our results, foreign protein localization in PB-I can be achieved by the temporal control of the different prolamin promoters.
-
Accumulation of foreign polypeptides to rice seed protein body type I using prolamin portion sequences.
Plant cell reports, 2016Co-Authors: Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Takehiro MasumuraAbstract:Rice Prolamins are accumulated in endoplasmic reticulum (ER)-derived proteins bodies, although conserved sequences retained in ER are not confirmed. We investigated portion sequences of Prolamins that must accumulate in PB-Is. Rice seed Prolamins are accumulated in endoplasmic reticulum (ER)-derived protein body type I (PB-I), but ER retention sequences in rice prolamin polypeptides have not been confirmed. Here we investigated the lengths of the prolamin portion sequences required for accumulation in PB-Is. Of the rice Prolamins, we compared 13a and 13b Prolamins because the amino acid sequences of these Prolamins are quite similar except for the presence or absence of Cys-residues. We also generated and analyzed transgenic rice expressing several prolamin portion sequence-GFP fusion proteins. We observed that in 13a prolamin, when the portion sequences were extended more than the 68th amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. In 13b prolamin, when the portion sequences were extended by more than the 82nd amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. When those fusion proteins were extracted under non-reduced or reduced conditions, the 13a prolamin portion sequence-GFP fusion proteins in PB-Is were soluble under only the reduced condition. In contrast, 13b prolamin portion sequence-GFP fusion proteins were soluble under both non-reduced and reduced conditions. These results suggest that the accumulation of 13a prolamin in PB-Is is associated with the formation of disulfide bonds and/or hydrophobicity in 13a prolamin polypeptide, whereas the accumulation of 13b prolamin in PB-Is was less involved in the formation of disulfide bonds.
-
Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system.
Bioscience biotechnology and biochemistry, 2014Co-Authors: Takehiro Masumura, Takanari Shigemitsu, Shigeto Morita, Shigeru SatohAbstract:Cereal Prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat Prolamins, rice Prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.
-
Accumulation of rice prolamin–GFP fusion proteins induces ER-derived protein bodies in transgenic rice calli
Plant cell reports, 2012Co-Authors: Takanari Shigemitsu, Takehiro Masumura, Shigeto Morita, Shigeru SatohAbstract:We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Rice Prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice Prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat Prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin–GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin–GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.
-
Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes
The Plant journal : for cell and molecular biology, 2012Co-Authors: Yuhi Saito, Kunisuke Tanaka, Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Ryuichi Yamasaki, Futami Goto, Koichi Kishida, Masaharu Kuroda, Shigeru SatohAbstract:Rice Prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice Prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa Prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b Prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.
