Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by nonvesicular mechanisms requiring carrier proteins. Oxysterol-binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for ergosterol transport in cells, indicating that they may be functionally redundant with each other or with additional-as yet unidentified-sterol carriers. To address this point, we hypothesized that sterol transport proteins are also sterol-binding proteins (SBPs), and used an in vitro chemoproteomic strategy to identify all cytosolic SBPs. We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analogue. Quantitative proteomics of the captured proteins identified 342 putative SBPs. Analysis of these identified proteins based on their annotated function, reported drug phenotypes, interactions with proteins regulating lipid metabolism, gene ontology, and presence of mammalian orthologues revealed a subset of 62 characterized and nine uncharacterized candidates. Five of the uncharacterized proteins play a role in maintaining plasma membrane integrity as their absence affects the ability of cells to grow in the presence of nystatin or myriocin. We anticipate that the dataset reported here will be a comprehensive resource for functional analysis of sterol-binding/transport proteins and provide insights into novel aspects of non-vesicular sterol trafficking.

中文翻译：

---

基于PhotoClick胆固醇的定量蛋白质组学筛查，用于酿酒酵母中的细胞质固醇结合蛋白。

麦角固醇是酵母质膜的重要组成部分，对酵母细胞的生存能力至关重要。它在内质网中合成，并通过需要载体蛋白的非囊泡机制转运至质膜。已经提出了氧固醇结合蛋白的同源物和酵母StARkin蛋白起固醇载体的作用。尽管这些蛋白质中的许多能够在体外在合成脂质囊泡之间转运固醇，但它们对于麦角固醇在细胞中的转运不是必需的，这表明它们彼此之间或与其他尚未鉴定的固醇载体在功能上可能是多余的。为了解决这一点，我们假设固醇转运蛋白也是固醇结合蛋白（SBPs），并使用体外化学旋转方法鉴定所有胞质SBP。我们产生了富含SBP的细胞溶质级分，并用光反应性可点击的胆固醇类似物捕获了蛋白质。捕获蛋白质的定量蛋白质组学确定了342个推定的SBP。根据其标注的功能，报告的药物表型，与调节脂质代谢的蛋白质的相互作用，基因本体论以及哺乳动物直系同源物的存在，对这些鉴定出的蛋白质进行分析，发现了62个特征化的候选者和9个未鉴定的候选者。未表征的蛋白质中有五个在维持质膜完整性方面起着作用，因为它们的缺失会影响在制霉菌素或myriocin存在下细胞的生长能力。