Shianna K V, Dotson W D, Tove S, Parks L W
Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27695-7615, USA.
J Bacteriol. 2001 Feb;183(3):830-4. doi: 10.1128/JB.183.3.830-834.2001.
Saccharomyces cerevisiae normally will not take up sterols from the environment under aerobic conditions. A specific mutant, upc2-1, of the predicted transcriptional activator UPC2 (YDR213w) has been recognized as a strain that allows a high level of aerobic sterol uptake. Another predicted transcriptional activator, the YLR228c gene product, is highly homologous to Upc2p. In fact, at the carboxy terminus 130 of the last 139 amino acids are similar between the two proteins. Since these proteins are very similar, the effect of mutations in the YLR228c open reading frame (ORF) was compared with like alterations in UPC2. First, the YLR228c ORF was insertionally inactivated and crossed with various UPC2 constructs. Deletion of YLR228c and UPC2 in combination resulted in nonviability, suggesting that the two proteins have some essential overlapping function. The upc2-1 point mutation responsible for aerobic sterol uptake was duplicated in the homologous carboxy region of the YLR228c ORF using site-directed mutagenesis. This mutation on a high-copy vector resulted in an increase in sterol uptake compared to an isogenic wild-type strain. The combination of both point mutations resulted in the greatest level of aerobic sterol uptake. When the YLR228c point mutation was expressed from a low-copy vector there was little if any effect on sterol uptake. Gas chromatographic analysis of the nonsaponifiable fractions of the various strains showed that the major sterol for all YLR228c and UPC2 combinations was ergosterol, the consensus yeast sterol.
在有氧条件下，酿酒酵母通常不会从环境中摄取固醇。预测的转录激活因子UPC2（YDR213w）的一个特定突变体upc2-1已被确认为一种能够进行高水平有氧固醇摄取的菌株。另一个预测的转录激活因子，即YLR228c基因产物，与Upc2p高度同源。事实上，在两种蛋白质的最后139个氨基酸的羧基末端130个氨基酸是相似的。由于这些蛋白质非常相似，因此将YLR228c开放阅读框（ORF）中的突变效应与UPC2中的类似改变进行了比较。首先，YLR228c ORF被插入失活，并与各种UPC2构建体杂交。YLR228c和UPC2的组合缺失导致细胞无法存活，这表明这两种蛋白质具有一些必需的重叠功能。使用定点诱变在YLR228c ORF的同源羧基区域复制了负责有氧固醇摄取的upc2-1点突变。与同基因野生型菌株相比，该高拷贝载体上的这种突变导致固醇摄取增加。两种点突变的组合导致了最高水平的有氧固醇摄取。当YLR228c点突变从低拷贝载体表达时，对固醇摄取几乎没有影响。对各种菌株的不皂化物部分进行气相色谱分析表明，所有YLR228c和UPC2组合的主要固醇都是麦角固醇，即公认的酵母固醇。
