Comparative proteome analysis of Saccharomyces cerevisiae grown in chemostat cultures limited for glucose or ethanol
Comparative proteome analysis of Saccharomyces cerevisiae grown in chemostat cultures limited for glucose or ethanol
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DOI：
10.1074/mcp.m400087-mcp200
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发表时间：
2005-01-01
影响因子：
7
通讯作者：
Slijper, M
中科院分区：
文献类型：
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作者：
Kolkman, A;Olsthoorn, MMA;Slijper, M
恒化培养的使用使得能够研究稳态生理特性和对营养限制生长的适应，而所有其他相关的生长条件保持不变。我们检测和比较了野生型酿酒酵母CEN的蛋白质组学反应。PK113-7D对有氧恒化器培养物生长的限制，碳源为葡萄糖或乙醇。为了获得蛋白质组变化的全球概况，我们使用二维凝胶电泳进行了三重分析，并使用MS鉴定了感兴趣的蛋白质。获得了大约400个蛋白质的相对数量，并进行了统计分析，以确定哪些蛋白质的稳定表达水平在葡萄糖或乙醇限制的条件下发生了显著变化。有趣的是，只有参与中心碳代谢的酶显示出显著的稳态表达变化，而这些酶中的15种只在两种碳源限制条件中的一种情况下检测到表达。没有观察到以前报道的分批培养条件下的副作用，如对特定生长率的连续变化、碳分解代谢抑制和有毒底物积累的反应。此外，通过将我们的蛋白质组数据与相应的mRNA数据进行比较，我们能够揭示中心碳代谢中的哪些过程在蛋白质组水平上受到调控，哪些过程在转录组水平上受到调控。重要的是，我们在这里展示了恒化培养和综合蛋白质组分析相结合的方法，使我们能够研究单一限制条件对酵母蛋白质组的主要影响。
The use of chemostat culturing enables investigation of steady-state physiological characteristics and adaptations to nutrient-limited growth, while all other relevant growth conditions are kept constant. We examined and compared the proteomic response of wild-type Saccharomyces cerevisiae CEN. PK113-7D to growth in aerobic chemostat cultures limited for carbon sources being either glucose or ethanol. To obtain a global overview of changes in the proteome, we performed triplicate analyses using two-dimensional gel electrophoresis and identified proteins of interest using MS. Relative quantities of about 400 proteins were obtained and analyzed statistically to determine which protein steady-state expression levels changed significantly under glucose- or ethanol-limited conditions. Interestingly, only enzymes involved in central carbon metabolism showed a significant change in steady-state expression, whereas expression was only detected in one of both carbon source-limiting conditions for 15 of these enzymes. Side effects that were previously reported for batch cultivation conditions, such as responses to continuous variation of specific growth rate, to carbon-catabolite repression, and to accumulation of toxic substrates, were not observed. Moreover, by comparing our proteome data with corresponding mRNA data, we were able to unravel which processes in the central carbon metabolism were regulated at the level of the proteome, and which processes at the level of transcriptome. Importantly, we show here that the combined approach of chemostat cultivation and comprehensive proteome analysis allowed us to study the primary effect of single limiting conditions on the yeast proteome.