Study of zein protein body formation in a heterologous system (Xenopus laevis oocyte)
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Most seed storage proteins accumulate in protein bodies which are derived from the vacuole. Zeins, the major corn storage proteins, however, are retained in the endoplasmic reticulum (ER) and their protein bodies are derived from the ER. There are circumstantial and preliminary data indicating that 27K zein, the proline-rich zein, may span the ER membrane. This potential transmembrane feature is considered very significant to understand the mechanism for zeins' ER retention. The transmembrane feature may retain the 27K zein in the ER where it could serve as an anchor for other classes of zein through specific protein interactions. In this study, a heterologous system (Xenopus laevis oocytes) was used to investigate the potential transmembrane domain of 27K zein. This study utilized physical assays of proteolytic digestion (proteinase K) and chemical modification (biotinylation) on isolated protein vesicles from Xenopus oocytes injected with in vitro transcribed 27K zein mRNA. In addition, the transmembrane features were analyzed by monitoring the protein's mobility in the lumen of the ER by pulse-chase experiments. The results showed that the possibility of 27K zein as a transmembrane protein was consistently refuted in this study. The 27K zein protein was not affected by the proteinase K treatment or biotinylation. Moreover, 27K zein and total zeins moved freely in the lumen of the ER similar to a secretory protein (ovalbumin), totally different from an ER membrane protein (a mutant transmembrane hemagglutinin envelope protein). The free movement, within the ER lumen, of total zeins under conditions where zein aggregates should form necessitates a reevaluation of the mechanisms responsible for zein polypeptides' ER retention and protein body formation. This study, therefore, concludes that 27K zein is not a protein body nucleating factor by virtue of an ER transmerrlbrane feature or association with the ER membrane and that the significance of zein solubility should be reconsidered to explain the zeins' ER retention leading to protein body formation in the ER.
- Doctoral Dissertations