Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Centre for Molecular Medicine and Therapeutics, British Columbia Children's Hospital Research Institute, University of British Columbia, Vancouver, Canada.
Elife. 2022 Nov 10;11:e74602. doi: 10.7554/eLife.74602.
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 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 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 (ipid-droplet rgosterol ortex 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.
细胞器膜之间的活跃维持的紧密毗邻，也称为接触位点，使生物分子在细胞隔室之间的有效转移成为可能。已经描述了几个这样的位点及其连接机制。尽管取得了这些进展，但我们仍然远未全面了解大多数接触位点的功能和调节。为了系统地表征接触位点蛋白质组，我们在基于共定位成像的背景下建立了一种高通量筛选方法。我们对六个不同接触位点的分裂荧光报告蛋白进行成像，其中一些是特征描述较差的，在背景下，表达了一个 mCherry 标记的酵母蛋白的 1165 个菌株，该蛋白具有细胞点状分布（接触位点的标志），受强 启动子的调节。通过对共定位事件和对报告蛋白大小和丰度的影响进行评分，我们在酵母中发现了 100 多个新的潜在接触点居民和效应物。我们专注于几个新鉴定的居民，鉴定了三个 Vps13 和 Atg2 的同源物，它们是多个接触位点的居民。这些蛋白质共享它们的脂质转运结构域，从而扩展了这个脂质转运蛋白家族。对另一个候选蛋白 Ypr097w 的分析，我们现在称之为 Lec1（ipid-droplet rgosterol ortex 1），揭示了这个以前未被表征的蛋白在脂质滴和细胞皮层之间动态移动，并在调节细胞中麦角固醇分布中发挥作用。总体而言，我们的分析扩展了接触点居民和效应物的范围，并创建了一个丰富的数据库，以挖掘新的功能、连接物和调节剂。
