LAUSR

A genetic screen in haploid human cells reveals context-specific regulators of the mTORC1 pathway. Perturb and observe screening in haploid cells Yeast have been a powerful model organism for genetic screens because they can easily be genetically manipulated during the haploid portion of their life cycle. De Zan et al. used a quantitative genetic screen in the HAP1 haploid human cell line that generated reproducible results in different laboratories without explicit standardization. The authors used this screening approach to identify genes whose loss of function by mutation increased or decreased the phosphorylation of ribosomal protein S6 (RPS6), a measure of the activity of mTORC1, a complex that mediates responses to nutrient availability. Adding pharmacological or genetic perturbations to the screen showed that LAMTOR4 enhanced the inhibition of mTORC1 by rapamycin, an effect not demonstrated by the related LAMTOR5, and that LAMTOR4 had a synthetic sick/lethal interaction with folliculin. This approach has the potential to reveal regulators of signaling pathways that function in specific contexts. Forward genetic screens in mammalian cell lines, such as RNAi and CRISPR-Cas9 screens, have made major contributions to the elucidation of diverse signaling pathways. Here, we exploited human haploid cells as a robust comparative screening platform and report a set of quantitative forward genetic screens for identifying regulatory mechanisms of mTORC1 signaling, a key growth control pathway that senses diverse metabolic states. Selected chemical and genetic perturbations in this screening platform, including rapamycin treatment and genetic ablation of the Ragulator subunit LAMTOR4, revealed the known core mTORC1 regulatory signaling complexes and the intimate interplay of the mTORC1 pathway with lysosomal function, validating the approach. In addition, we identified a differential requirement for LAMTOR4 and LAMTOR5 in regulating the mTORC1 pathway under fed and starved conditions. Furthermore, we uncovered a previously unknown “synthetic-sick” interaction between the tumor suppressor folliculin and LAMTOR4, which may have therapeutic implications in cancer treatment. Together, our study demonstrates the use of iterative “perturb and observe” genetic screens to uncover regulatory mechanisms driving complex mammalian signaling networks.