RUI: Endoplasmic Reticulum and Protein Secretion in Heat Shocked Plant Cells

RUI：热休克植物细胞中的内质网和蛋白质分泌

• 批准号: 9105888
• 负责人: Mark Brodl
• 金额: $ 23.6万
• 依托单位: Knox College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1995-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9105888&HistoricalAwards=false
• 关键词: RUI; Endoplasmic; Reticulum; Protein; Secretion

The long-term goal of this research is to understand the cell biology of higher plant responses to environmental stress. The model system for investigation is the heat shock response of the aleurone layer of barley grains. The cells of this tissue are normally dedicated to protein secretion; however, heat shock dramatically redirects their cellular activities. The synthesis of secretory proteins is abruptly arrested when the tissue is subjected to heat shock, yet the synthesis of nonsecretory proteins continues. This is accomplished by the selective destabilization of otherwise stable secretory protein mRNAs. Heat shock also causes the loss of endoplasmic reticulum lamellar structures upon which secretory protein mRNAs are translated; only small fragments of ER remain. This appears to provide the discriminatory mechanism for selectively stopping the synthesis of secretory proteins, as nonsecretory protein mRNAs are not translated by ER-bound ribosomes. The work proposed investigates the molecular and cellular basis for these heat shock-induced events. In vitro translation reactions supplemented with barley microsomes will be used to investigate whether the ER fragments of heat-shocked aleurone cells can serve as sites for the translation and translocation of proteins. The ER lamellae are supported by a cytoskeleton. In other organisms, the cytoskeleton has been shown to be unstable during heat shock. Immunofluorescence microscopy will be used to investigate the effect of heat shock on cytoskeletal elements of barley aleurone cells. Heat-induced shifts in the buoyant densities of ER membranes have been observed; these will be investigated by assaying protein:lipid ratios of microsomes isolated on sucrose density gradients. Secretory protein mRNAs contain secretory signals that direct them to be translated on ER lamellae. Aleurone cells transformed with signal sequence - reporter gene constructs will be used to determine of the presence of such a signal is sufficient to render the mRNA unstable during heat shock. These experiments should provide insights into the cell biology of ER, as well as reveal mechanisms for regulating protein expression during heat shock. These efforts will ultimately build a better understanding of how plants respond and adapt to stress.

本研究的长期目标是了解高等植物对环境胁迫的细胞生物学反应。研究的模型系统是大麦籽粒糊粉层的热激响应。这种组织的细胞通常专门用于蛋白质分泌；然而，热休克戏剧性地改变了它们的细胞活动。当组织受到热休克时，分泌性蛋白质的合成突然停止，而非分泌性蛋白质的合成仍在继续。这是通过选择性地破坏其他稳定的分泌蛋白mrna来实现的。热休克还会导致内质网板层结构的丧失，而分泌蛋白mrna是在内质网板层结构上被翻译的；只剩下少量的ER片段。这似乎提供了选择性停止分泌蛋白合成的歧视性机制，因为非分泌蛋白mrna不被er结合的核糖体翻译。所提出的工作研究了这些热冲击诱导事件的分子和细胞基础。在体外翻译反应中补充大麦微粒体将用于研究热休克糊粉细胞的内质网片段是否可以作为蛋白质翻译和易位的位点。内质网片由细胞骨架支撑。在其他生物体中，细胞骨架已被证明在热休克期间是不稳定的。利用免疫荧光显微镜研究热休克对大麦糊粉细胞骨架成分的影响。热引起的内质网膜浮力密度的变化已经被观察到；这些将通过分析在蔗糖密度梯度上分离的微粒体的蛋白：脂质比率来研究。分泌蛋白mrna含有指示它们在内质网片上翻译的分泌信号。用信号序列报告基因构建转化的糊粉细胞将被用来确定这种信号的存在是否足以使mRNA在热休克期间不稳定。这些实验将有助于深入了解内质网的细胞生物学，并揭示热休克过程中调节蛋白表达的机制。这些努力最终将有助于更好地理解植物如何应对和适应压力。