Blomberg A
Göteborg University, Sweden.
Electrophoresis. 1997 Aug;18(8):1429-40. doi: 10.1002/elps.1150180818.
Cells respond to increased external osmolarities by enhanced accumulation of compatible solutes. In yeast-cells, mainly exemplified by Saccharomyces cerevisiae, the premier compatible solute is the polyhydroxy-alcohol glycerol, the production of which is accompanied by overall metabolic changes. By applying two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled to computerized image quantification, a large body of valuable physiological information relating to this stress-adaptation has been gathered. One of the presumed key-enzymes in the production of glycerol in the cell is glycerol 3-phosphate dehydrogenase encoded by the GPD1 gene. The amount of this protein is enhanced during saline stress, and from 2-D analysis linked to microsequencing it became apparent that the osmo-regulated from contained a putative presequence. Sequence analysis of another salt-induced spot in the 2-D pattern revealed identity to a gene, YER062c, with previously unknown function. Biochemical characterization of this protein, including standard purification via chromatography and subsequent activity/specificity measurements, identified this salt-regulated protein as the missing protein/gene in glycerol production, namely the glycerol 3-phosphatase. The sequence of another salt regulated protein resolved in the 2-D gel revealed identity to a bacterial dihydroxyacetone kinase, thus indicating salt induced glycerol dissimilation. Comparing Northern data to the 2-D generated expression pattern revealed a strong correlation, indicating mainly regulation at the transcriptional level. In addition, altered expression during saline growth of some of the glycolytic enzymes was also apparent. Signalling mutants, either in the cAMP-dependent protein kinase A pathway or in a protein kinase cascade, have been analyzed during osmotic stress via 2-D PAGE, grouping proteins/genes apparently regulated via similar mechanismus. Proteome analysis has proven invaluable in the unravelling of the molecular physiology of yeast cells during adaptation and growth under osmotic stress, identifying vital components not selected by purely genetic approaches.
细胞通过增强相容性溶质的积累来应对外部渗透压的升高。在以酿酒酵母为主要代表的酵母细胞中，首要的相容性溶质是多羟基醇甘油，其产生伴随着整体代谢变化。通过应用二维聚丙烯酰胺凝胶电泳（2-D PAGE）结合计算机图像定量分析，已经收集了大量与这种应激适应相关的有价值的生理信息。细胞中甘油产生过程中推测的关键酶之一是由GPD1基因编码的甘油3-磷酸脱氢酶。在盐胁迫期间，这种蛋白质的量会增加，并且通过与微量测序相关的二维分析表明，渗透压调节的蛋白含有一个推定的前序列。二维图谱中另一个盐诱导斑点的序列分析显示与一个功能未知的基因YER062c相同。对该蛋白质的生化特性进行表征，包括通过色谱法进行标准纯化以及随后的活性/特异性测量，确定这种盐调节蛋白是甘油产生中缺失的蛋白质/基因，即甘油3-磷酸酶。在二维凝胶中解析的另一种盐调节蛋白的序列显示与一种细菌二羟基丙酮激酶相同，从而表明盐诱导的甘油异化作用。将Northern数据与二维生成的表达模式进行比较，发现有很强的相关性，表明主要是在转录水平上进行调节。此外，一些糖酵解酶在盐胁迫生长期间的表达变化也很明显。已经通过二维聚丙烯酰胺凝胶电泳分析了在渗透胁迫期间cAMP依赖性蛋白激酶A途径或蛋白激酶级联中的信号突变体，将明显通过类似机制调节的蛋白质/基因进行了分组。蛋白质组分析已被证明在揭示酵母细胞在渗透胁迫下的适应和生长过程中的分子生理学方面具有重要价值，它鉴定出了一些通过纯遗传方法未筛选到的重要成分。
