Candida albicans TDH3 gene promotes secretion of internal invertase when expressed in Saccharomyces cerevisiae as a glyceraidehyde-3-phosphate dehydrogenase-invertase fusion protein
Candida albicans TDH3 gene promotes secretion of internal invertase when expressed in Saccharomyces cerevisiae as a glyceraidehyde-3-phosphate dehydrogenase-invertase fusion protein
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作者：
Delgado, ML;Gil, ML;Gozalbo, D
我们通过使用细胞内形式的酵母转化酶作为报告蛋白，检查了白色念珠菌 GAPDH 多肽（缺乏常规 N 末端信号肽）到达酿酒酵母细胞壁的能力。在着丝粒衍生质粒中构建杂合TDH3-SUC2基因，该杂合TDH3-SUC2基因含有白色念珠菌TDH3启动子序列和编码由白色念珠菌GAPDH多肽形成的融合蛋白的编码区，在其C末端与酵母内部转化酶融合，并转化到Suc(-)酿酒酵母菌株中。在完整的全细胞中测量到转化体表现出蔗糖酶活性，并且能够以蔗糖作为唯一的可发酵碳源生长。使用 TDH3 和 SUC2 探针进行的 Northern 印迹分析检测到预期大小（约 2.7 kb）的单一 mRNA 种类，使用针对白色念珠菌 GAPDH 表位的单克隆抗体 (mAb49) 对无细胞提取物进行蛋白质印迹分析，结果显示存在与 GAPDH-转化酶融合蛋白相对应的 90 kDa 多肽。这表明 TDH3 基因能够将部分编码基因产物引导至细胞壁，并且用于该靶向的任何推定基序应位于 GAPDH 氨基酸序列内。使用相同的方法，对七个 N 端和 C 端 GAPDH 截短物进行进一步分析，结果表明细胞壁靶向所需的区域位于蛋白质的 N 端一半内。版权所有 (C) 2003 John Wiley Sons, Ltd.
We have checked the ability of the Candida albicans GAPDH polypeptide, which lacks a conventional N-terminal signal peptide, to reach the cell wall in Saccharomyces cerevisiae by using an intracellular form of the yeast invertase as a reporter protein. A hybrid TDH3-SUC2 gene containing the C albicans TDH3 promoter sequences and a coding region encoding a fusion protein formed by the C albicans GAPDH polypeptide, fused at its C-terminus with the yeast internal invertase, was constructed in a centromer derivative plasmid and transformed into a Suc(-) S. cerevisiae strain. Transformants displayed invertase activity measured in intact whole cells, and were able to grow on sucrose as the sole fermentable carbon source. Northern blot analysis with both TDH3 and SUC2 probes detected a single mRNA species of the expected size (about 2.7 kb), and Western immunoblot analysis of cell-free extracts, using a monoclonal antibody (mAb49) against a C. albicans GAPDH epitope, showed the presence of a 90 kDa polypeptide corresponding to the GAPDH-invertase fusion protein. This indicates that the TDH3 gene is able to direct part of the encoded gene product to the cell wall, and that any putative motifs for this targeting should be within the GAPDH amino acid sequence. Further analysis, using the same approach, of a panel of seven N- and C-terminal GAPDH truncates revealed that the region required for the cell wall targeting is located within the N-terminal half of the protein. Copyright (C) 2003 John Wiley Sons, Ltd.