This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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