Leung James, Karachaliou Mayia, Alves Claudia, Diallinas George, Byrne Bernadette
Division of Molecular Biology, Imperial College London, South Kensington, London SW7 2AZ, UK.
Protein Expr Purif. 2010 Jul;72(1):139-46. doi: 10.1016/j.pep.2010.02.002. Epub 2010 Feb 11.
The Nucleobase-Ascorbate Transporters (NATs) family includes carriers with fundamental functions in uptake of key cellular metabolites, such as uric acid or vitamin C. The best studied example of a NAT transporter is the uric acid-xanthine permease (UapA) from the model ascomycete Aspergillus nidulans. Detailed genetic and biochemical analyses have revealed much about the mechanism of action of this protein; however, the difficulties associated with handling eukaryotic membrane proteins have limited efforts to elucidate the precise structure-function relationships of UapA by structural analysis. In this manuscript, we describe the heterologous overexpression of functional UapA as a fusion with GFP in different strains of Saccharomyces cerevisiae. The UapA-GFP construct expressed to 2.3 mg/L in a pep4Delta deletion strain lacking a key vacuolar endopeptidase and 3.8 mg/L in an npi1-1 mutant strain with defective Rsp5 ubiquitin ligase activity. Epifluorescence microscopy revealed that the UapA-GFP was predominately localized to the plasma membrane in both strains, although a higher intensity of fluorescence was observed for the npi1-1 mutant strain plasma membrane. In agreement with these observations, the npi1-1 mutant strain demonstrated a approximately 5-fold increase in uptake of [(3)H]-xanthine compared to the pep4Delta deletion strain. Despite yielding the best results for functional expression, in-gel fluorescence of the UapA-GFP expressed in the npi1-1 mutant strain revealed that the protein was subject to significant proteolytic degradation. Large scale expression of the protein using the pep4Delta deletion strain followed by purification produced mg quantities of pure, monodispersed protein suitable for further structural and functional studies. In addition, this work has generated a yeast cell based system for performing reverse genetics and other targeted approaches, in order to further understand the mechanism of action of this important model protein.
核碱基 - 抗坏血酸转运蛋白（NATs）家族包括在摄取关键细胞代谢物（如尿酸或维生素C）方面具有基本功能的载体。NAT转运蛋白中研究得最透彻的例子是来自模式子囊菌构巢曲霉的尿酸 - 黄嘌呤通透酶（UapA）。详细的遗传和生化分析已经揭示了该蛋白的许多作用机制；然而，处理真核膜蛋白所面临的困难限制了通过结构分析阐明UapA精确结构 - 功能关系的努力。在本手稿中，我们描述了功能性UapA与GFP融合在不同酿酒酵母菌株中的异源过表达。UapA - GFP构建体在缺乏关键液泡内肽酶的pep4Δ缺失菌株中表达量为2.3 mg/L，在具有缺陷的Rsp5泛素连接酶活性的npi1 - 1突变菌株中表达量为3.8 mg/L。落射荧光显微镜显示，在这两种菌株中，UapA - GFP主要定位于质膜，尽管在npi1 - 1突变菌株的质膜上观察到更高强度的荧光。与这些观察结果一致，与pep4Δ缺失菌株相比，npi1 - 1突变菌株对[³H] - 黄嘌呤的摄取增加了约5倍。尽管在功能表达方面产生了最佳结果，但在npi1 - 1突变菌株中表达的UapA - GFP的凝胶内荧光显示该蛋白受到显著的蛋白水解降解。使用pep4Δ缺失菌株大规模表达该蛋白，随后进行纯化，产生了毫克量的纯的、单分散的蛋白，适合进一步的结构和功能研究。此外，这项工作已经产生了一个基于酵母细胞的系统，用于进行反向遗传学和其他靶向方法，以便进一步了解这种重要模式蛋白的作用机制。
