Our current understanding of mitochondrial organelle physiology has benefited from two broad approaches: classically, cuvette-based measurements with suspensions of isolated mitochondria, in which bioenergetic parameters are monitored acutely in response to respiratory chain substrates and inhibitors^1,2,3,4, and more recently, highly scalable genetic screens for fitness phenotypes associated with coarse-grained properties of the mitochondrial state^5,6,7,8,9,10. Here we introduce permeabilized-cell mitochondrial function sequencing (PMF-seq) to combine strengths of these two approaches to connect genes to detailed bioenergetic phenotypes. In PMF-seq, the plasma membranes within a pool of CRISPR mutagenized cells are gently permeabilized under conditions that preserve mitochondrial physiology, where detailed bioenergetics can be probed in the same way as with isolated organelles. Cells with desired bioenergetic parameters are selected optically using flow cytometry and subjected to next-generation sequencing. Using PMF-seq, we recover genes differentially required for mitochondrial respiratory chain branching and reversibility. We demonstrate that human d-lactate dehydrogenase specifically conveys electrons from d-lactate into cytochrome c to support mitochondrial membrane polarization. Finally, we screen for genetic modifiers of tBID, a pro-apoptotic protein that acts directly and acutely on mitochondria. We find the loss of the complex V assembly factor ATPAF2 acts as a genetic sensitizer of tBID’s acute action. We anticipate that PMF-seq will be valuable for defining genes critical to the physiology of mitochondria and other organelles.

我们目前对线粒体细胞器生理学的理解受益于两种广泛的方法：传统上，使用分离的线粒体悬浮液进行基于比色皿的测量，其中根据呼吸链底物和抑制剂^1,2,3,4敏锐地监测生物能参数，以及最近，针对与线粒体状态粗粒度特性相关的健康表型进行了高度可扩展的遗传筛选^5,6,7,8,9,10。在这里，我们引入透化细胞线粒体功能测序 (PMF-seq)，结合这两种方法的优势，将基因与详细的生物能表型联系起来。在 PMF-seq 中，CRISPR 诱变细胞库中的质膜在保留线粒体生理学的条件下被温和地透化，其中可以以与分离的细胞器相同的方式探测详细的生物能学。使用流式细胞术光学选择具有所需生物能参数的细胞并进行下一代测序。使用 PMF-seq，我们恢复了线粒体呼吸链分支和可逆性所需的差异基因。我们证明人d-乳酸脱氢酶特异性地将电子从d-乳酸传递到细胞色素c以支持线粒体膜极化。最后，我们筛选了 tBID 的遗传修饰剂，tBID 是一种直接、急性作用于线粒体的促凋亡蛋白。我们发现复合体 V 组装因子 ATPAF2 的缺失可作为 tBID 急性作用的遗传敏化剂。我们预计 PMF-seq 对于定义对线粒体和其他细胞器生理学至关重要的基因非常有价值。