IDENTIFYING REPLICATIVE AGE-INDUCED CHANGES IN S CEREVISIAE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 发芽酵母具有有限的复制能力，被用作细胞老化的模型。复制寿命被定义为母细胞衰老之前母细胞产生的子细胞的数量。由于培养中的绝大多数细胞都是年轻的，在其复制寿命接近尾声时，很难获得大量的老细胞。丰富复制衰老母细胞的遗传系统的发展使我们能够在复制寿命接近尾声的时候净化大量的细胞。 我们已经进行了全局表达分析，以确定年轻细胞和老年细胞之间的差异。在衰老细胞中上调的基因富含细胞壁的结构成分和在细胞壁生物发生中起作用的基因，这表明在衰老细胞中细胞壁完整性途径被激活。为了补充这些发现，我们已经开始采取一种公正的方法来识别复制的老细胞和年轻细胞的蛋白质组的差异。我们将与华盛顿大学的迈克尔·麦考斯合作，通过质谱学确定蛋白质丰度的任何定量差异。 从衰老细胞和年轻细胞中提取的全细胞蛋白提取物的SDS-PAGE分析表明，在衰老细胞中有大量的高分子量物种(&gt；175 kDa)。对这些物种的质谱分析表明，它们富含内质网膜蛋白，并且预测它们的相对分子质量比它们的SDS-PAGE迁移所显示的要低得多。因此，它们很可能是翻译后修饰的，可能是通过糖基化。 我们推测，在衰老细胞中，蛋白质在内质网和/或高尔基复合体中发生了异常的糖基化。这种缺陷可能导致分泌途径的缺陷，并触发细胞壁完整性途径。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Budding yeast has a finite replicative capacity and is used as a model for cellular aging. Replicative life span is defined as the number of daughter cells produced by a mother cell before the mother cell senesces. Because the vast majority of cells in culture are young, it is very difficult to obtain large quantities of old cells near the end of their replicative lifespan. The development of a genetic system that enriches for replicatively aged mother cells has allowed us to purify significant numbers of cells near the end of their replicative life span. We have performed global expression analysis to identify differences between young and old cells. Upregulated genes in old cells are enriched for structural components of the cell wall and genes that function in cell wall biogenesis, suggesting that the cell wall integrity pathway is activated in old cells. To complement these findings, we have begun to take an unbiased approach to identify differences in the proteome of replicatively old and young cells. We will identify any quantitative differences in protein abundance by mass spectrometry in collaboration with Michael MacCoss at the University of Washington. SDS-PAGE analysis of whole-cell protein extract from old and young cells reveals an enrichment of high molecular weight species (>175 kDa) specifically in old cells. Mass spectrometry analyses of these species indicate that they are enriched for endoplasmic reticulum membrane proteins and are predicted to have substantially lower molecular weights than suggested by their SDS-PAGE migration. It is therefore likely that they are post-translationally modified, perhaps by glycosylation. We speculate that proteins are aberrantly glycosylated in the endoplasmic reticulum and/or golgi complex in old cells. Such a defect could lead to deficiencies in the secretory pathway and trigger the cell wall integrity pathway.