Sonawala Unnati, Busidan Aymeric, Haak David, Pilot Guillaume
School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia, United States of America.
PLoS One. 2025 Apr 30;20(4):e0315789. doi: 10.1371/journal.pone.0315789. eCollection 2025.
Saccharomyces cerevisiae mutants have been used since the early 1980s as a tool for characterizing genes from other organisms by functional complementation. This approach has been extremely successful in cloning and studying transporters; for instance, plant amino acid, sugar, urea, ammonium, peptide, sodium, and potassium transporters were characterized using yeast mutants lacking these functions. Over the years, new strains lacking even more endogenous transporters have been developed, enabling the characterization of transport properties of heterologous proteins in a more precise way. Furthermore, these strains provide the added possibility of characterizing a transporter belonging to a family of proteins in isolation, and thus can be used to study the relative contribution of redundant transporters to the whole function. We focused on amino acid transport, starting with the yeast strain 22 ∆ 8AA, which was developed to clone plant amino acid transporters in the early 2000s. We recently deleted two additional amino acid permeases, Gnp1 and Agp1, creating 22 ∆ 10α. In the present work, five additional permeases (Bap3, Tat1, Tat2, Agp3, Bap2) were deleted from 22 ∆ 10α genome, in a combination of up to three at a time. Unexpectedly, the amino acid transport properties of the new strains were not very different from the parent, suggesting that these amino acid permeases play a minor role in amino acid uptake, at least in our conditions. Furthermore, the inability to utilize certain amino acids as sole nitrogen source did not correlate with reduced uptake activity, questioning the well-accepted relationship between lack of growth and loss of transport properties. Finally, in order to verify the mutations and the integrity of 22 ∆ 10α genome, we performed whole-genome sequencing of 22 ∆ 10α using long-read PacBio sequencing technology. We successfully assembled 22 ∆ 10α's genome de novo, identified all expected mutations and precisely characterized the nature of the deletions of the ten amino acid transporters. The sequencing data and genome will serve as a valuable resource to researchers interested in using these strains as a tool for amino acid transport study.
自20世纪80年代初以来，酿酒酵母突变体就被用作通过功能互补来表征其他生物体基因的工具。这种方法在克隆和研究转运蛋白方面极其成功；例如，利用缺乏这些功能的酵母突变体对植物氨基酸、糖、尿素、铵、肽、钠和钾转运蛋白进行了表征。多年来，已经开发出了缺乏更多内源性转运蛋白的新菌株，从而能够更精确地表征异源蛋白的转运特性。此外，这些菌株还提供了单独表征属于一个蛋白质家族的转运蛋白的可能性，因此可用于研究冗余转运蛋白对整体功能的相对贡献。我们从21世纪初开发用于克隆植物氨基酸转运蛋白的酵母菌株22 ∆ 8AA开始，专注于氨基酸转运研究。我们最近又删除了另外两个氨基酸通透酶Gnp1和Agp1，构建了22 ∆ 10α。在本研究中，从22 ∆ 10α基因组中一次最多删除三个另外的通透酶（Bap3、Tat1、Tat2、Agp3、Bap2）。出乎意料的是，新菌株的氨基酸转运特性与亲本菌株并没有太大差异，这表明这些氨基酸通透酶在氨基酸摄取中起的作用较小，至少在我们的实验条件下是这样。此外，无法利用某些氨基酸作为唯一氮源与摄取活性降低并无关联，这对普遍接受的生长缺乏与转运特性丧失之间的关系提出了质疑。最后，为了验证22 ∆ 10α基因组的突变和完整性，我们使用长读长PacBio测序技术对22 ∆ 10α进行了全基因组测序。我们成功地从头组装了22 ∆ 10α的基因组，鉴定出所有预期的突变，并精确表征了十个氨基酸转运蛋白缺失的性质。测序数据和基因组将为有兴趣将这些菌株用作氨基酸转运研究工具的研究人员提供宝贵的资源。
