Mitochondrial and ER membrane protein trafficking CRISPR screens

The trafficking of specific protein cohorts to the correct subcellular
location at the correct time 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 FACS. To broadly 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
identify genes that influence the trafficking of mitochondrial and ER
tail-anchored proteins. We show that loss of the SUMO E1 component SAE1
results in the mislocalization and destabilization of 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.