An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens

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. 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.

Determinants of trafficking, conduction, and disease within a K+ channel revealed through multiparametric deep mutational scanning

“…Many other assay types are compatible with this approach, providing opportunities for even richer phenotypic description of ion channel variation. For example, a recently described approach, HiLITR ( Coukos et al, 2021 ), could provide more granular resolution about trafficking motifs and Kir2.1 localization in living cells. Spontaneously-spiking HEK cells ( Hochbaum et al, 2014 ; Park et al, 2013 ), which co-express Kir2.1, a voltage-dependent Na + channel (Nav1.7), the channelrhodopsin CheRiff, and genetically encoded voltage indicator QuasAr2, could be adapted to evaluate how ion channel expression levels and gating properties impact excitability ( O’Leary et al, 2014 ).…”

MTCH2 is a mitochondrial outer membrane protein insertase

“…These results are consistent with the EMC serving as the major insertase for mislocalized mitochondrial TAs into the ER (fig. S4) ( 11 ), and the UBQLNs’ role in degrading mislocalized mitochondrial TAs ( 12 ), leading to their accumulation in the cytosol (fig. S5).…”

MTCH2 is a mitochondrial outer membrane protein insertase

“…Similarly, knockdown of the ER membrane protein complex (EMC), which we found was the major pathway for insertion of mislocalized mitochondrial TAs into the ER, also increases insertion into the mitochondria in agreement with recent studies (fig. S4) (17). Presumably, depletion of either the UBQLNs or the EMC increases the cytosolic pool of OMP25, allowing additional opportunities for successful insertion into the outer membrane prior to degradation by the ubiquitin proteasome pathway.…”

“…3F). As a positive control, we also reconstituted the human EMC, which readily mis-inserts mitochondrial TAs (fig S11) (17), using a purification (25) and reconstitution strategy (26) as previously described. In a protease protection assay, OMP25 synthesized in native cytosol was inserted into MTCH2-containing proteoliposomes in a dose-dependent manner that correlated with MTCH2 concentration (Fig.…”