在酿酒酵母中,已经描述了两种类型的海藻糖酶活性。中性海藻糖酶(Nth1和Nth2)被认为是催化细胞内海藻糖动员的主要蛋白质。除Nth1和Nth2外,研究表明,酸性海藻糖酶Ath1是细胞外海藻糖降解所必需的。尽管已经在酿酒酵母的实验室菌株中主要研究了中性和酸性海藻糖酶,我们试图研究破坏野生菌株中这些基因的表型后果。在这项研究中,我们在5个不同的酿酒酵母菌株中构建了海藻糖降解途径(NTH1,NTH2和ATH1)的突变体,以检查已发表的实验室菌株的表型是否也由野生菌株表现出来.对于每个突变体,我们评估了许多表型,以与海藻糖生物合成突变体进行比较。包括海藻糖生产,糖原生产,细胞大小,急性耐热性,高温生长,孢子形成效率,以及在丰富和基本培养基中的各种碳源上的生长。我们发现所有的海藻糖酶突变体包括单个缺失nth1Δ,nth2Δ,和ath1Δ,与它们的等基因野生型细胞相比,双缺失nth1nth2Δ积累了更高的细胞内海藻糖水平。此外,nth1Δ和nth1Δnth2Δ突变体表现出轻度的热敏感性,提示当细胞从压力中恢复时,海藻糖动员的潜在次要作用。此外,我们评估了与海藻糖降解更直接相关的表型,包括细胞外和细胞内海藻糖的利用。我们发现细胞内海藻糖水解对于典型的孢子萌发进程至关重要,强调海藻糖在细胞周期调节中的作用,可能作为一种储存碳水化合物提供糖酵解燃料。此外,我们的工作提供了进一步的证据,表明Ath1对于细胞外海藻糖作为碳源的利用是必不可少的,即使在AGT1的存在。
In the yeast Saccharomyces cerevisiae, two types of
trehalase activities have been described. Neutral trehalases (Nth1 and Nth2) are considered to be the main proteins that catalyze intracellular trehalose mobilization. In addition to Nth1 and Nth2, studies have shown that acid
trehalase Ath1 is required for extracellular trehalose degradation. Although both neutral and acid-type trehalases have been predominantly investigated in laboratory strains of S. cerevisiae, we sought to examine the phenotypic consequences of disrupting these genes in wild strains. In this study, we constructed mutants of the trehalose degradation pathway (NTH1, NTH2, and ATH1) in five diverse S. cerevisiae strains to examine whether published lab strain phenotypes are also exhibited by wild strains. For each mutant we assessed a number of phenotypes for comparison to trehalose biosynthesis mutants, including trehalose production, glycogen production, cell size, acute thermotolerance, high temperature growth, sporulation efficiency, and growth on a variety of carbon sources in rich and minimal medium. We found that all
trehalase mutants including single deletion nth1Δ, nth2Δ, and ath1Δ, as well as double deletion nth1nth2Δ accumulated higher intracellular trehalose levels compared to their isogenic wild type cells. Also, nth1Δ and nth1Δnth2Δ mutants exhibited mild thermal sensitivity, suggesting a potential minor role for trehalose mobilization when cells recover from stress. In addition, we evaluated phenotypes more directly relevant to trehalose degradation, including both extracellular and intracellular trehalose utilization. We discovered that intracellular trehalose hydrolysis is critical for typical spore germination progression, highlighting a role for trehalose in cell cycle regulation, likely as a storage carbohydrate providing glycolytic fuel. Additionally, our work provides further evidence suggesting Ath1 is indispensable for extracellular trehalose utilization as a carbon source, even in the presence of AGT1.