Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution.
Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution.
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DOI：
10.7554/elife.74602
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发表时间：
2022-11-10
期刊：
影响因子：
7.7
通讯作者：
Schuldiner M
中科院分区：
文献类型：
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作者：
Castro IG;Shortill SP;Dziurdzik SK;Cadou A;Ganesan S;Valenti R;David Y;Davey M;Mattes C;Thomas FB;Avraham RE;Meyer H;Fadel A;Fenech EJ;Ernst R;Zaremberg V;Levine TP;Stefan C;Conibear E;Schuldiner M
细胞器膜之间主动保持紧密并置，也称为接触位点，使得生物分子能够在细胞区室之间有效转移。已经描述了几个这样的网站以及它们的拴系机械。尽管取得了这些进展，我们仍然远远没有全面了解大多数接触部位的功能和调节。为了系统地表征接触位点蛋白质组，我们建立了一种基于共定位成像的酿酒酵母高通量筛选方法。我们成像分裂荧光记者为6个不同的接触点，其中几个是不好的特点，在1165株表达mCherry标记的酵母蛋白，具有细胞点状分布（接触点的标志）的背景下，调节强TEF2启动子。通过对共定位事件和对报告分子大小和丰度的影响进行评分，我们在酵母中发现了100多个新的潜在接触位点居民和效应子。集中在几个新发现的居民，我们确定了三个同源的Vps13和Atg2的居民的多个接触点。这些蛋白质共享它们的脂质转运结构域，从而扩展了脂质转运蛋白家族。对另一个候选者Ypr097w（我们现在称之为Lec1（脂滴麦角固醇皮质1））的分析表明，这种以前未表征的蛋白质在脂滴和细胞皮质之间动态转移，并在调节细胞中麦角固醇分布中发挥作用。总的来说，我们的分析扩展了接触点居民和效应器的范围，并创建了一个丰富的数据库来挖掘新的功能，系绳和监管机构。
Actively maintained close appositions between organelle membranes, also known as contact sites, enable the efficient transfer of biomolecules between cellular compartments. Several such sites have been described as well as their tethering machineries. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize contact site proteomes, we established a high-throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, several of which are poorly characterized, on the background of 1165 strains expressing a mCherry-tagged yeast protein that has a cellular punctate distribution (a hallmark of contact sites), under regulation of the strong TEF2 promoter. By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified three homologs of Vps13 and Atg2 that are residents of multiple contact sites. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell. Overall, our analysis expands the universe of contact site residents and effectors and creates a rich database to mine for new functions, tethers, and regulators.