Abstract. Wheat seed storage proteins are deposited in protein bodies (PB) inside vacuoles, but their subcellular site of aggregation and their route to vacuoles are still controversial. In the present work, an ultra structural analysis of developing wheat endosperm at early to mid maturation was performed to address these issues. Golgi complexes were rarely detected, indicating that their role in wheat storage protein transport is limited. In contrast, a considerable amount of PB was detected in the cytoplasm. Many of these PB were surrounded by PER membranes and were enlarged by fusion of smaller PB. Small, electron lucent vesicles were detected around the surfaces of the PB in the cytoplasm, or attached to them, suggesting that such attachments and subsequent fusion of the vesicles with each other lead to the formation of small vacuoles containing PB inclusions. Immunogold labeling with serum raised against yeast-BiP, an ER-localized protein, demonstrated that the wheat BiP homolog was present within the PB in the cytoplasm as well as inside vacuoles. This confirmed that the PB were formed within the RER and that the Golgi complex was not involved in their transport to vacuoles. It is concluded that a considerable part of the wheat storage proteins aggregate into PB within the RER and are then transported as intact PB to the vacuoles by a novel route that does not utilize the Golgi complex.
Storage proteins of wheat grains (Triticum L. em Thell) are deposited in protein bodies inside vacuoles. However, the subcellular sites and mechanisms of their aggregation into protein bodies are not clear. In the present report, we provide evidence for two different types of protein bodies, low-and high-density types that accumulate concurrently and independently in developing wheat endosperm cells. Gliadins were present in both types of protein bodies, whereas the high molecular weight glutenins were localized mainly in the dense ones. Pulse-chase experiments verified that the dense protein bodies were not formed by a gradual increase in density but, presumably, by a distinct, quick process of storage protein aggregation. Subcellular fractionation and electron microscopy studies revealed that the wheat homolog of immunoglobulin heavy-chain-binding protein, an endoplasmic reticulum-resident protein, was present within the dense protein bodies, implying that these were formed by aggregation of storage proteins within the endoplasmic reticulum. The present results suggest that a large part of wheat storage proteins aggregate into protein bodies within the rough endoplasmic reticulum. Because these protein bodies are too large to enter the Golgi, they are likely to be transported directly to vacuoles. This route may operate in concert with the known Golgi-mediated transport to vacuoles in which the storage proteins apparently condense into protein bodies at a postendoplasmic reticulum location. Our results further suggest that although gliadins are transported by either one of these routes, the high molecular weight glutenins use only the Golgi bypass route.
[alpha]-Gliadins and [gamma]-gliadins are two closely related wheat storage proteins that evolved from a common ancestral gene. However, synthesis of [alpha]-gliadins and [gamma]-gliadins in Xenopus laevis oocytes revealed striking differences in their subcellular routing. The major portion of [alpha]-gliadin accumulated inside the oocyte, whereas most of the [gamma]-gliadin was secreted. Disruption of the Golgi apparatus by monensin revealed that the major part of secretion of [gamma]-gliadin is Golgi mediated. The difference in the subcellular route between [alpha]-gliadin and [gamma]-gliadin may be attributed to differential transport from the endoplasmic reticulum to the Golgi apparatus, a process that is generally the rate-limiting step in protein secretion. Coinjection of the two mRNAs had no effect on their routing, indicating no interaction between them. Our results support the hypothesis that subcellular transport of gliadins in wheat endosperm occurs in two separate routes; one is Golgi mediated, and the other is not. We also show that the subcellular transport may be markedly affected by small structural variations within closely related storage proteins.
[alpha]-Gliadins and [gamma]-gliadins are two closely related wheat storage proteins that evolved from a common ancestral gene. However, synthesis of [alpha]-gliadins and [gamma]-gliadins in Xenopus laevis oocytes revealed striking differences in their subcellular routing. The major portion of [alpha]-gliadin accumulated inside the oocyte, whereas most of the [gamma]-gliadin was secreted. Disruption of the Golgi apparatus by monensin revealed that the major part of secretion of [gamma]-gliadin is Golgi mediated. The difference in the subcellular route between [alpha]-gliadin and [gamma]-gliadin may be attributed to differential transport from the endoplasmic reticulum to the Golgi apparatus, a process that is generally the rate-limiting step in protein secretion. Coinjection of the two mRNAs had no effect on their routing, indicating no interaction between them. Our results support the hypothesis that subcellular transport of gliadins in wheat endosperm occurs in two separate routes; one is Golgi mediated, and the other is not. We also show that the subcellular transport may be markedly affected by small structural variations within closely related storage proteins.
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