AN MF ALPHA-1-SUC2 (ALPHA-FACTOR-INVERTASE) GENE FUSION FOR STUDY OF PROTEIN LOCALIZATION AND GENE-EXPRESSION IN YEAST
AN MF ALPHA-1-SUC2 (ALPHA-FACTOR-INVERTASE) GENE FUSION FOR STUDY OF PROTEIN LOCALIZATION AND GENE-EXPRESSION IN YEAST
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DOI：
10.1073/pnas.80.23.7080
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
THORNER, J
中科院分区：
文献类型：
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作者：
EMR, SD;SCHEKMAN, R;THORNER, J
肽交配信息素。-因子和水解酶转化酶。- d -果糖呋喃苷果糖水解酶（EC 3.2.1.26）是从酵母细胞（酿酒酵母）分泌过程中由较大的前体蛋白加工而成。通过连接α编码序列的5”侧区和前导体部分，构建了两种蛋白结构基因的框架内融合。-因子基因（MF.alpha.1）与转化酶基因（SUC2）的一个大片段（缺少其5''-侧翼区域及其信号序列的前4个氨基酸的编码信息）相关联。与正常蛋白水解过程有关的位点。-因子前驱体保留在该结构中。嵌合基因指导高水平的活性转化酶的合成，这种酶被有效地分泌到质周空间，允许细胞在含蔗糖的培养基上生长。这种细胞外转化酶似乎不含pre - α。因素序列。然而，最初的细胞内产物是一种杂交蛋白，可以通过用药物tunicamycin处理细胞或通过阻断温度条件下分泌缺陷突变体的分泌来检测（sec18）。因此，在有效的蛋白水解去除之前，. α。杂交蛋白的-因子部分显然为更大的蛋白转化酶的有效输出提供了必要的信息。类似于MF.alpha。1, mf。1-SUC2融合在。alpha中表达。单倍体的水平比单倍体高65-75倍。二倍体;此外，在mat.alpha中消除了高级表达式。1个突变体，但不是mat。alpha。2突变体。与SUC2的表达不同，融合蛋白的表达不受葡萄糖浓度的影响。因此，融合中存在的5”侧区（约950个碱基对）足以产生α。杂交基因的细胞特异性表达。
The peptide mating pheromone .alpha.-factor and the hydrolytic enzyme invertase (.beta.-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (S. cerevisiae). An in-frame fusion of the structural genes for these 2 proteins was constructed by connecting the 5''-flanking region and preproleader portion of the coding sequence of the .alpha.-factor gene (MF.alpha.1) to a large fragment of the invertase gene (SUC2) lacking its 5''-flanking region and the coding information for the first 4 amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the .alpha.-factor precursor were retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-.alpha.-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18). Therefore, prior to its efficient proteolytic removal, the .alpha.-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MF.alpha.1, the MF.alpha.1-SUC2 fusion is expressed in .alpha. haploids at levels 65-75 times higher than in a haploids or in a/.alpha. diploids; also, high-level expression is eliminated in mat.alpha.1 mutants but not in mat.alpha.2 mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5''-flanking region present in the fusion (about 950 base pairs) is sufficient to confer .alpha. cell-specific expression to the hybrid gene.