Fur4-mediated uracil-scavenging to screen for surface protein regulators.

IF 3.6 3区生物学 Q3 CELL BIOLOGY

Traffic Pub Date : 2021-11-01 Epub Date: 2021-09-23 DOI:10.1111/tra.12815

Katherine M Paine, Gabrielle B Ecclestone, Chris MacDonald

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引用次数: 2

Abstract

Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the uracil permease Fur4 in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a library of haploid deletion strains and identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multisubunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterized genes. Bioinformatic analysis of expression profiles from many strains lacking transcription factors required for efficient uracil-scavenging validated particular hits from the screen, in addition to implicating essential genes not tested in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorize factors implicated in uracil-scavenging.

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Fur4介导的尿嘧啶清除以筛选表面蛋白调节因子。

细胞表面膜蛋白具有多种关键功能，并受膜运输途径的时空调节。尽管这些通路中的扰动是许多病理的基础，但我们对这些通路的机制水平的理解仍然不完整。以酵母为模型，我们开发了一种检测方法，报告了在有限尿嘧啶存在下生长的尿嘧啶营养不良菌株中尿嘧啶渗透酶Fur4的表面活性。该试验用于筛选单倍体缺失菌株文库，并鉴定出在限制性尿嘧啶培养基中相对生长减弱和增强的突变体。所鉴定的因子，包括各种多亚基复合物，除了各种未表征的基因外，还富集了膜运输和转录功能。对许多缺乏有效清除尿嘧啶所需转录因子的菌株的表达谱进行生物信息学分析，验证了筛选中的特定命中，以及筛选中未测试的必要基因。最后，我们进行了次级交配因子分泌筛选，以对与尿清除有关的因子进行功能分类。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Traffic 生物-细胞生物学

CiteScore

8.10

自引率

2.20%

发文量

审稿时长

2 months

期刊介绍： Traffic encourages and facilitates the publication of papers in any field relating to intracellular transport in health and disease. Traffic papers span disciplines such as developmental biology, neuroscience, innate and adaptive immunity, epithelial cell biology, intracellular pathogens and host-pathogen interactions, among others using any eukaryotic model system. Areas of particular interest include protein, nucleic acid and lipid traffic, molecular motors, intracellular pathogens, intracellular proteolysis, nuclear import and export, cytokinesis and the cell cycle, the interface between signaling and trafficking or localization, protein translocation, the cell biology of adaptive an innate immunity, organelle biogenesis, metabolism, cell polarity and organization, and organelle movement. All aspects of the structural, molecular biology, biochemistry, genetics, morphology, intracellular signaling and relationship to hereditary or infectious diseases will be covered. Manuscripts must provide a clear conceptual or mechanistic advance. The editors will reject papers that require major changes, including addition of significant experimental data or other significant revision. Traffic will consider manuscripts of any length, but encourages authors to limit their papers to 16 typeset pages or less.