Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology

We thank J. Nunnari and M. Le Vasseur for sharing the mitochondrial split GFP system, and J.M. Replogle for helping establish the CRISPRi screening system using the split GFP strategy. We thank Y.H. Chen for assistance with computational analyses, and Z. Levine and K.E. Yost for careful reading and input on the manuscript. We thank the Whitehead Institute and Koch Institute Flow Cytometry Cores for access to FACS machines, the Whitehead Institute Genome Technology Core for support with sequencing of screen libraries, the Whitehead Institute W.M. Keck Microscopy Facility for access to confocal microscopes and support with imaging analysis, and the Whitehead Proteomics Core for support for mass spectrometry experiments. This work was supported by the NIH pre-doctoral training grant T32GM007287 (G.M.), the Larry L. Hillblom Foundation (A.J.I.), the Fannie and John Hertz Foundation and NSF Graduate Research Fellowships (R.A.S.), the Heritage Medical Research Institute (R.M.V.), the Human Frontier Science Program 2019L/LT000858 (A.G.), and the Howard Hughes Medical Institute (J.S.W.). Models in Figures 4 and 7 and the graphical abstract were created with the help of BioRender.

G.M., A.G., and J.S.W. were responsible for the conception, design, and interpretation of experiments, and wrote the manuscript with input from all authors. G.M. led all in-cell experiments and accompanying data analysis with help from T.K.E., who performed all RT-qPCR experiments and assisted with flow cytometry and immunoprecipitation studies. T.A.S., A.J.I., and A.G. led all in vitro experiments and performed accompanying data analysis, with supervision from R.M.V. R.A.S. performed analysis of genome-scale perturb-seq data. F.S. led all mass spectrometry experiments and accompanying data analysis. J.S.W. and R.M.V. funded the study.

• CRISPRi genome-scale screen gene-level phenotypes are available in Table S1. Mass spectrometry data are available in Table S3. Original western blot images have been deposited at Mendeley and are publicly available as of the date of publication. The DOI is listed in the key resources table and here: https://data.mendeley.com/preview/xtbb222g5w?a=eb24a7b8-52f8-4f10-aa5f-a26b7c1af0eb (final https://doi.org/10.17632/xtbb222g5w.1). Microscopy data reported in this paper will be shared by the lead contact upon request.
• This paper does not report any original code.
• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

J.S.W. declares outside interest in 5 AM Venture, Amgen, Chroma Medicine, KSQ Therapeutics, Maze Therapeutics, Tenaya Therapeutics, Tessera Therapeutics, and Third Rock Ventures. R.M.V. is a consultant and equity holder in Gate Bioscience. J.S.W. is a member of the Molecular Cell advisory board.