Lubkowitz Mark A, Hauser Loren, Breslav Michael, Naider Fred, Becker Jeffrey M
Department of Microbiology and Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, TN 37996-0845, USA.
University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-8080, USA.
Microbiology (Reading). 1997 Feb;143 ( Pt 2):387-396. doi: 10.1099/00221287-143-2-387.
A Candida albicans oligopeptide transport gene, OPT1, was cloned from a C. albicans genomic library through heterologous expression in the Saccharomyces cerevisiae di-/tripeptide transport mutant PB1X-9B. When transformed with a plasmid harbouring OPT1, S. cerevisiae PB1X-9B, which did not express tetra-/pentapeptide transport activity under the conditions used, was conferred with an oligopeptide transport phenotype, as indicated by growth on the tetrapeptide Lys-Leu-Leu-Gly, sensitivity to toxic tetra- and pentapeptides, and an increase in the initial uptake rate of the radiolabelled tetrapeptide Lys-Leu-Gly-[3H]Leu. The level of oligopeptide transport was found to be influenced in the heterologous host by the source of nitrogen used for growth. The entire 3.8 kb fragment containing the oligopeptide transport activity was sequenced and an ORF of 2349 nucleotides containing a 58 nucleotide intron was identified. The deduced protein product of 783 amino acid residues contained 12 hydrophobic regions suggestive of a membrane transport protein. Sequence comparisons revealed that similar proteins are encoded by genes from S. cerevisiae and Schizosaccharomyces pombe and that OPT1 is not a member of the ABC or PTR membrane transport families.
通过在酿酒酵母二肽/三肽转运突变体PB1X - 9B中的异源表达,从白色念珠菌基因组文库中克隆出一个白色念珠菌寡肽转运基因OPT1。用携带OPT1的质粒转化时,在所用条件下不表达四肽/五肽转运活性的酿酒酵母PB1X - 9B获得了寡肽转运表型,这通过在四肽Lys - Leu - Leu - Gly上生长、对有毒四肽和五肽的敏感性以及放射性标记的四肽Lys - Leu - Gly - [³H]Leu初始摄取速率的增加得以表明。发现寡肽转运水平在异源宿主中受用于生长的氮源影响。对包含寡肽转运活性的整个3.8 kb片段进行测序,鉴定出一个含有58个核苷酸内含子的2349个核苷酸的开放阅读框。推导的由783个氨基酸残基组成的蛋白质产物含有12个疏水区域,提示其为膜转运蛋白。序列比较显示,酿酒酵母和粟酒裂殖酵母的基因编码相似的蛋白质,且OPT1不是ABC或PTR膜转运家族的成员。