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

这项研究的长期目标是了解高等植物对环境胁迫的反应的细胞生物学。研究的模式系统是大麦籽粒糊粉层的热激响应。这种组织的细胞通常致力于蛋白质的分泌；然而，热休克会戏剧性地改变它们的细胞活动。当组织受到热休克时，分泌性蛋白质的合成突然停止，但非分泌性蛋白质的合成仍在继续。这是通过选择性地使其他稳定的分泌蛋白mRNAs失稳来实现的。热休克还会导致内质网层片状结构的丧失，分泌蛋白mRNAs在内质网层状结构上翻译；只有一小部分内质网残存。这似乎为选择性地停止分泌蛋白的合成提供了歧视性机制，因为非分泌蛋白mRNAs不被内质网结合的核糖体翻译。这项工作旨在研究这些热休克诱导事件的分子和细胞基础。在添加大麦微体的体外翻译反应中，将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。内质网片层由细胞骨架支撑。在其他生物体中，细胞骨架已被证明在热休克期间不稳定。免疫荧光显微镜将用于研究热休克对大麦糊粉细胞细胞骨架元素的影响。已经观察到了热诱导的ER膜浮力密度的变化；这将通过分析蔗糖密度梯度上分离的微体的蛋白质：脂比来进行研究。分泌蛋白mRNAs含有分泌信号，指导它们在内质网片层上被翻译。用信号序列报告基因结构转化的糊粉细胞将被用来确定这样的信号的存在足以使mrna在热休克期间不稳定。这些实验应该提供对内质网细胞生物学的洞察，以及揭示热休克期间调节蛋白质表达的机制。这些努力最终将建立对植物如何应对和适应压力的更好理解。

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.