Abstract
The trafficking of specific protein cohorts to correct subcellular locations at correct times is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or fluorescence-activated cell sorting (FACS). To empower the study of protein trafficking processes with gene perturbation, we developed a genetically encoded molecular tool named HiLITR (High-throughput Localization Indicator with Transcriptional Readout). HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identified genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in mislocalization and destabilization of many mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.
Highlights
Pooled screens simplify the handling of large libraries with >105 unique elements (Kampmann et al, 2015; Morgens et al, 2016; Wang et al, 2015), but require that the cellular function of interest be coupled to a simple readout, such as cell proliferation (Han et al, 2020; Kory et al, 2018) or FACS (Dejesus et al, 2016; Potting et al, 2017)
We selected SAE1 (Fig. 4) and seven additional hits from Figures 3D/E for validation. Four of these hits (SAE1, CCNK, SKA1, and ATP6V1A) gave robust validation (Figure 4 and S8E), and we found by imaging that knockdown of SKA1 or ATP6V1A increased the fraction of GFP-mTA* protease mislocalized to the Golgi (Fig. S8F/G)
The biology which can be accessed by pooled-format screens is most limited by our ability to couple a cellular function of interest to a simple, robust readout
Summary
To combine the strengths of the pooled screen format (library size, simplicity) and the arrayed screen format (versatility in readout), we sought to develop a molecular reporter capable of converting complex cellular processes such as protein trafficking or mislocalization into simple single-timepoint, intensity-based FACS readouts. Such a tool would enable screening of large libraries in a pooled format without sacrificing the versatility and specificity required to probe more complex cellular processes. To design HiLITR, we required a mechanism to convert protein localization or mislocalization in live cells to a simple readout for pooled genetic screens.
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