Vollert, M. | Search bioRxiv

bioRxiv2025

Genetic identification of the RAS proteostatic machinery and its failure to regulate oncogenic variants

The regulation of cellular homeostasis, differentiation, and proliferation is safeguarded by proteostatic mechanisms, which are crucial for maintaining cellular function. We used endogenously affinity-tagged KRAS cells in a fluorescence-activated cell sorting (FACS)-based CRISPR knockout screen to map genome-wide genetic requirements for proteostatic KRAS regulation. Three regulatory modules emerged, specifically cullin E3 ligase activity (CUL3, NAE1, UBE2M, CAND1, and UBE2L3), LZTR1 protein function (LZTR1, NUDCD3, and ZRSR2), and RAS GTPase modulation and processing (NRAS, HRAS, FNTB, RCE1, ICMT, and GOLGA7), all critical for regulating KRAS abundance. This expands our knowledge on the machinery controlling ubiquitin-meditated RAS family member regulation. Combining endogenous affinity tagging with genetic variant introduction, we found that the oncogenic KRAS G12D mutant exhibits reduced regulation by CRL3LZTR1, potentially contributing to oncogenic transformation and proliferation. In summary, our study genetically defines the machinery regulating RAS GTPase protein abundance, providing a foundation for a deeper molecular understanding and potential therapeutic exploitation of CRL3LZTR1-RAS GTPase regulation in human disease.

cell biology↗

bioRxiv2022

Gain-of-function genetic screens in human cells identify SLC transporters overcoming environmental nutrient restrictions

Solute carrier (SLC) transporters control fluxes of nutrients and metabolites across membranes and thereby represent a critical interface between the microenvironment and cellular and subcellular metabolism. Because of substantial functional overlap, the interplay and relative contributions of members of this family in response to environmental stresses remain poorly elucidated. In order to infer functional relationships between SLCs and metabolites, we developed a strategy to identify human SLCs able to sustain cell viability and proliferation under growth-limiting concentrations of essential nutrients. One-by-one depletion of 13 amino acids required for cell proliferation enabled gain-of-function genetic screens using a SLC-focused CRISPR/Cas9-based transcriptional activation approach to uncover transporters relieving cells from the growth-limiting metabolic bottleneck. We identified the cationic amino acid transporter SLC7A3 as a gene that, when upregulated, overcame low availability of arginine and lysine by increasing their uptake. SLC7A5 (LAT1), on the other hand, was able to sustain cellular fitness upon deprivation of several neutral amino acids. A genome-wide screen identified SLC7A3 as the single main gene product able to rescue cell survival in the limiting arginine conditions tested, demonstrating the potentially decisive role of transporters in overcoming nutrient limitations. Moreover, we identified metabolic compensation mediated by the glutamate/aspartate transporters SLC1A2 and SLC1A3 under glutamine-limiting conditions. Overall, this gain-of-function approach using human cells led to the definition of functional transporter-nutrient relationships and revealed that upregulation of transport activity may be sufficient to overcome environmental metabolic restrictions.

cell biology↗