An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens
- Department of Genetics, Stanford University, United States
- Chan Zuckerberg Biohub, United States
- Broad Institute of MIT and Harvard, United States
- Whitehead Institute, United States
- Department of Biology, Massachusetts Institute of Technology, United States
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, United States
- Howard Hughes Medical Institute, University of California, San Francisco, United States
- Department of Biology, Stanford University, United States
Figures
Figure 1 with 5 supplements
HiLITR gives transcriptional readout of protein localization in living cells.
(A) Schematic of HiLITR. HiLITR has two components: a low-affinity protease (green) and a membrane-anchored transcription factor (TF, red). Left: when protease and TF are colocalized on the same …
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Figure 1—source data 1
Source data for Figure 1F.
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Details for HiLITR constructs used in this study.
LOV, TF, and TEV protease domains used in Figure 1—figure supplements 2 and 3 (HiLITR optimization) vary slightly. Any differences are shown and discussed in the text.
Figure 1—figure supplement 2
Sequential optimization of HiLITR components.
(A) Fluorescence-activated cell sorting (FACS) plots of HEK cells transiently transfected with unoptimized HiLITR components. Transcription factor (TF) is on the outer mitochondrial membrane (OMM), …
Figure 1—figure supplement 3
Optimization of HiLITR experimental parameters.
(A) Fluorescence-activated cell sorting (FACS) plots of K562 cells stably expressing optimized mitochondrial HiLITR components (outer mitochondrial membrane [OMM]-targeted protease and transcription …
Figure 1—figure supplement 4
Additional characterization of HiLITR constructs and cell lines.
(A) Immunofluorescence microscopy of stably-integrated HiLITR components used in fluorescence-activated cell sorting (FACS) experiment in Figure 1D. The localizations of the mitochondrial …
Figure 1—figure supplement 5
Model selection on K562 cells expressing mitochondrial transcription factor (TF) HiLITR.
Same as Figure 1E and F, except that cells expressing mitochondrial protease (colocalized with TF) are combined with cells expressing ER protease (rather than cytosolic protease as in Figure 1E). …
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Figure 1—figure supplement 5—source data 1
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig1-figsupp5-data1-v2.xlsx
Figure 2 with 1 supplement
HiLITR reads out protein mislocalization or loss in CRISPRi screens.
(A) Possible outcomes for sgRNA disruption of mitochondrial protease in cells expressing mitochondrial HiLITR transcription factor (TF) and protease. In the first example, sgRNA #1 disrupts protease …
Figure 2—figure supplement 1
Whole-genome CRISPRi screen with HiLITR readout.
(A) CasTLE plot showing results of whole-genome CRISPRi screen, performed in two replicates in the clonal K562 mito transcription factor (TF)/mito protease HiLITR cell line (shown in Figure 2C, …
Figure 3 with 4 supplements
CRISPRi screen with HiLITR readout identifies proteins that influence the localization of mitochondrial membrane and ER membrane proteins.
(A) CasTLE plot showing combined results from tail-anchored (TA), signal-anchored (SA), and ER CRISPRi screens. x-axis plots the TA screen score (lower when the mito TA protease from Figure 2C is …
Figure 3—figure supplement 1
Retesting of transmembrane recognition complex (TRC)/GET pathway genes with HiLITR.
(A) The plot from Figure 3A, with genes in the TRC pathway (GET pathway in yeast) labeled. All five genes tested in the sublibrary screens (WRB and CAMLG were omitted) produced significant increases …
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Figure 3—figure supplement 1—source data 1
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig3-figsupp1-data1-v2.xlsx
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Figure 3—figure supplement 1—source data 2
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig3-figsupp1-data2-v2.xlsx
Figure 3—figure supplement 2
Retesting individual sgRNAs from the three HiLITR configuration CRISPRi screen.
(A) Fluorescence-activated cell sorting (FACS) plots showing the effects of individual sgRNAs on HiLITR readout in three K562 HiLITR cell lines (tail-anchored [TA], signal-anchored [SA], and ER) …
Figure 3—figure supplement 3
Retesting individual sgRNAs in polyclonal cell lines.
(A) Fluorescence-activated cell sorting (FACS) plots showing the effects of individual sgRNAs on HiLITR readout in polyclonal K562 HiLITR cell lines (corresponding to tail-anchored [TA], …
Figure 3—figure supplement 4
Analysis of other pathways in CRISPRi screening data.
Figure 4 with 1 supplement
SAE1 knockdown disrupts localization and abundance of mitochondrial tail-anchored (TA) proteins.
(A) SAE1 knockdown by CRISPRi reduces HiLITR activation in TA screen configuration, while increasing HiLITR activation in SA and ER screen configurations. (B) Quantitation of data in (A). Log2-transf…
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Figure 4—source data 1
Source data for Figure 4E.
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig4-data1-v2.xlsx
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Figure 4—source data 2
Source data for Figure 4G.
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig4-data2-v2.xlsx
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Figure 4—source data 3
Source data for Figure 4I.
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig4-data3-v2.xlsx
Figure 4—figure supplement 1
Additional data on SAE1.
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4 - figure supplement 1E.
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig4-figsupp1-data1-v2.xlsx
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Figure 4—figure supplement 1—source data 2
Source data for Figure 4 - figure supplement 1J.
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig4-figsupp1-data2-v2.xlsx
Figure 5 with 5 supplements
SAE1 knockdown reduces the abundance of many endogenous mitochondrial tail-anchored (TA) proteins.
(A) HeLa cells infected with SAE1 sgRNA or nontargeting control for 9 days were analyzed by western blot. Three TA mitochondrial proteins (MAVS, SYNJ2BP, FIS1) were analyzed in addition to three …
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Figure 5—source data 1
Source data for Figure 5B.
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Uncropped western blots used to make Figure 5A and B.
Data from sgRNA #2 (blue) was used to generate Figure 4A (boxed regions).
Figure 5—figure supplement 2
Western blots of SAE1 knockdown and rescue.
HeLa cells expressing dCas9-KRAB were infected for 9 days with either nontargeting control (NT) or sgRNA against SAE1. Each sample was further transduced with either sgRNA-resistant SAE1 or …
Figure 5—figure supplement 3
Whole-proteome profiling data.
(A) Overview of proteomic experiment. HeLa cells expressing either sgRNA-resistant SAE1 or nonfluorescent BFP (nfBFP) were infected with either nontargeting control sgRNA or sgRNA against SAE1 for 9 …
Figure 5—figure supplement 4
Mitochondrial proteome data normalized to mean mitochondrial protein abundance.
(A) Same as Figure 5C, but zoomed out to show the entire volcano plot. HeLa cells expressing nonfluorescent BFP (nfBFP) were infected with sgRNA against SAE1 (‘SAE1 knockdown’) or with nontargeting …
Figure 5—figure supplement 5
ER proteome data normalized to mean ER protein abundance.
(A) Knockdown of SAE1 does not significantly affect ER tail-anchored protein abundance relative to other ER proteins. HeLa cells expressing nonfluorescent BFP and sgRNA against SAE1 (‘SAE1 …
Figure 6 with 2 supplements
EMC10 has opposite regulatory effect on ER tail-anchored (TA) proteins as other ER membrane complex (EMC) subunits.
(A) Locations of 9 of 10 EMC components in the 3-CRISPRi screen CasTLE plot from Figure 3A. EMC5 was not included in the screen. In the table at right, corresponding effect sizes from each screen …
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Figure 6—source data 1
Source data for Figure 6D.
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig6-data1-v2.xlsx
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Figure 6—source data 2
Source data for Figure 6F.
- https://cdn.elifesciences.org/articles/69142/elife-69142-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
Additional HiLITR analysis related to the ER membrane complex (EMC).
(A) Fluorescence-activated cell sorting (FACS) analysis related to Figure 6B. Three HiLITR configurations (as in Figure 2C–E) in K562 cells with sgRNAs against EMC subunits and two nontargeting …
Figure 6—figure supplement 2
Uncropped western blots used to make Figure 6E and F.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | HEK293T | ATCC | Cat# CRL-3216; RRID:CVCL_0063 | |
| Cell line (human) | K562 | ATCC | Cat# CCL-243; RRID:CVCL_0004 | |
| Cell line (human) | HeLa | Hein et al., 2015 | RRID:CVCL_1922 | |
| Antibody | Anti-V5 (Mouse monoclonal) | Invitrogen | Cat# R960; RRID:AB_2556564 | Immunofluorescence (1:1000) |
| Antibody | Anti-TOMM20 (Rabbit monoclonal) | Abcam | Cat# ab186735; RRID:AB_2889972 | Immunofluorescence (1:500) |
| Antibody | Anti-GRASP65 (Mouse monoclonal) | Santa Cruz | Cat# sc-374423; RRID:AB_10991322 | Immunofluorescence (1:500) |
| Antibody | Anti-CANX (Rabbit polyclonal) | Thermo Fisher | Cat# PA5-34754; RRID:AB_2552106 | Immunofluorescence (1:500) |
| Antibody | Anti-PEX14 (Rabbit polyclonal) | Proteintech | Cat# 10594-1-AP; RRID:AB_2252194 | Immunofluorescence (1:500) |
| Antibody | Anti-RCN2 (Rabbit polyclonal) | Thermo Fisher | Cat# PA5-56542; RRID:AB_2646431 | Immunofluorescence (1:500) |
| Antibody | Anti-GAPDH (Mouse monoclonal) | Santa Cruz | Cat# sc-32233; RRID:AB_627679 | Western blot (1:4500) |
| Antibody | Anti-SAE1 (Rabbit polyclonal) | Sigma | Cat# SAB4500028; RRID:AB_10742679 | Western blot (1:500) |
| Antibody | Anti-COX4 (Rabbit polyclonal) | Abcam | Cat# ab16056; RRID:AB_443304 | Western blot (1:1000) |
| Antibody | Anti-VDAC1 (Mouse monoclonal) | Abcam | Cat# ab14734; RRID:AB_443084 | Western blot (1:500) |
| Antibody | Anti-AKAP1 (Mouse monoclonal) | Santa Cruz | Cat# sc-135824; RRID:AB_2225573 | Immunofluorescence (1:200); western blot (1:500) |
| Antibody | Anti-MAVS (Mouse monoclonal) | Santa Cruz | Cat# sc-166583; RRID:AB_2012300 | Immunofluorescence (1:200); western blot (1:250) |
| Antibody | Anti-SYNJ2BP (Rabbit polyclonal) | Sigma | Cat# HPA000866; RRID:AB_2276678 | Western blot (1:500) |
| Antibody | Anti-FIS1 (Rabbit polyclonal) | Thermo Fisher | Cat# 10956–1-AP; RRID:AB_2102532 | Western blot (1:1000) |
| Antibody | Anti-EMC4 (Rabbit monoclonal) | Abcam | Cat# ab184162; RRID:AB_2801471 | Western blot (1:1000) |
| Antibody | Anti-EMC10 (Rabbit monoclonal) | Abcam | Cat# ab180148; RRID:AB_2889936 | Western blot (1:500) |
| Antibody | Anti-CALR (Rabbit polyclonal) | Thermo Fisher | Cat# PA3900; RRID:AB_325990 | Western blot (1:500) |
| Antibody | Anti-VTI1B (Rabbit monoclonal) | Abcam | Cat# ab184170; RRID:AB_2889935 | Western blot (1:250) |
| Antibody | Anti-SQS (Rabbit monoclonal) | Abcam | Cat# ab195046; RRID:AB_2860018 | Western blot (1:500) |
| Antibody | Anti-Mouse Alexa Fluor 488 (Goat polyclonal) | Invitrogen | Cat#: A11029; RRID:AB_138404 | Immunofluorescence (1:1000) |
| Antibody | Anti-Mouse Alexa Fluor 568 (Goat polyclonal) | Invitrogen | Cat# A11031; RRID:AB_144696 | Immunofluorescence (1:1000) |
| Antibody | Anti-Mouse Alexa Fluor 647 (Goat polyclonal) | Invitrogen | Cat#: A21236; RRID:AB_2535805 | Immunofluorescence (1:1000) |
| Antibody | Anti-Rabbit Alexa Fluor 568 (Goat polyclonal) | Invitrogen | Cat#: A11036; RRID:AB_10563566 | Immunofluorescence (1:1000) |
| Antibody | Anti-Rabbit Alexa Fluor 405 (Goat polyclonal) | Invitrogen | Cat# A31556; RRID:AB_221605 | Immunofluorescence (1:1000) |
| Antibody | Anti-Mouse IgG IRDye 680RD (Goat polyclonal) | Licor | Cat# 926-68070; RRID:AB_10956588 | Western blot (1:20,000) |
| Antibody | Anti-Mouse IgG IRDye 800CW (Goat polyclonal) | Licor | Cat# 926-32210; RRID:AB_621842 | Western blot (1:20,000) |
| Antibody | Anti-Rabbit IgG IRDye 680RD (Goat polyclonal) | Licor | Cat# 926-68071; RRID:AB_10956166 | Western blot (1:20,000) |
| Antibody | Anti-Rabbit IgG IRDye 800CW (Goat polyclonal) | Licor | Cat# 926-32211; RRID:AB_621843 | Western blot (1:20,000) |
| Recombinant DNA reagent | Plasmids used | This paper | N/A | Supplementary file 1 |
| Sequence-based reagent | HiLITR TEV-protease QPCR primers | This paper | N/A | Materials and methods: ‘Model selection’ |
| Sequence-based reagent | Random hexamer primer | Invitrogen | Cat# N8080127 | |
| Sequence-based reagent | Individual sgRNA sequences used | This paper | N/A | Supplementary file 1 |
| Sequence-based reagent | sgRNA libraries derived from hCRISPRi-v2 | Horlbeck et al., 2016 | RRID:Addgene_83969 | Supplementary file 2 |
| Peptide, recombinant protein | Fibronectin | Millipore | Cat# FC010 | |
| Peptide, recombinant protein | Bovine serum albumin | Fisher BioReagents | Cat# BP1600 | |
| Peptide, recombinant protein | Aprotinin | Sigma | Cat# A1153 | |
| Peptide, recombinant protein | Leupeptin | Roche | Cat# 11017101001 | |
| Peptide, recombinant protein | Endoproteinase LysC | Wako Laboratories | Cat# 12505061 | |
| Peptide, recombinant protein | Sequencing-grade trypsin | Promega | Cat# V5111 | |
| Commercial assay or kit | RNeasy Plus Mini Kit | Qiagen | Cat# 74134 | |
| Commercial assay or kit | QIAamp DNA Blood Maxi Kit | Qiagen | Cat# 51194 | |
| Commercial assay or kit | BCA Assay Kit | Pierce | Cat# 23225 | |
| Commercial assay or kit | MycoAlert Mycoplasma detection kit | Lonza | Cat# LT07-118 | |
| Chemical compound, drug | 1% penicillin-streptomycin | Corning | Cat# 30-002CI | |
| Chemical compound, drug | GlutaMAX | Gibco | Cat# 35050061 | |
| Chemical compound, drug | Puromycin | Sigma | Cat# P8833 | |
| Chemical compound, drug | Blasticidin | Corning | Cat# 30-100-RB | |
| Chemical compound, drug | Hygromycin | Corning | Cat# 30-240-CR | |
| Chemical compound, drug | Geneticin G418 | Thermo Fisher | Cat# 10131035 | |
| Chemical compound, drug | Polyethyleneimine (PEI) | Polysciences | Cat# 24765-1 | |
| Chemical compound, drug | Polybrene | Millipore | Cat# TR-1003-G | |
| Chemical compound, drug | Doxycycline | Sigma | Cat# C9891 | |
| Chemical compound, drug | MitoTracker Deep Red FM | Invitrogen | Cat# M22426 | |
| Chemical compound, drug | Paraformaldehyde | RICCA | Cat# 3180 | |
| Chemical compound, drug | Triton X-100 | Sigma | Cat# T9284 | |
| Chemical compound, drug | TMTpro isobaric mass tagging reagent | Thermo | Cat# A44520 | |
| Software, algorithm | CasTLE | Morgens et al., 2016 | https://bitbucket.org/dmorgens/castle/src | |
| Software, algorithm | Bowtie 2 | Langmead and Salzberg, 2012 | RRID::SCR_016368 | |
| Software, algorithm | SH800S Cell Sorter Software (versions 2.1.2, 2.1.5) | SONY | N/A | |
| Software, algorithm | Everest (version 2.3) | BioRad | N/A | |
| Software, algorithm | FlowJo (version 10.7.1) | FlowJo | N/A | |
| Software, algorithm | SlideBook 5.0 software | Intelligent Imaging Innovations | N/A | |
| Software, algorithm | StepOne Software (version 2.2.2) | Applied Biosystems | N/A | |
| Software, algorithm | Limma (version 3.42.2) | Smyth, 2004 | RRID:SCR_010943 | |
| Other | Fetal bovine serum | Avantor | Cat# 97068-085 | |
| Other | SuperScript III Reverse Transcriptase | Invitrogen | Cat# 18080093 | |
| Other | RiboLock RNAse inhibitor | Thermo Scientific | Cat# EO0382 | |
| Other | Maxima SYBR Green/ROX qPCR Master Mix | Thermo Scientific | Cat# K0221 | |
| Other | Herculase II Fusion | Agilent | Cat# 600679 | |
| Other | Protease Inhibitor Cocktail | Sigma | Cat# P8849 | |
| Other | Precision Plus Protein All Blue Prestained Standards | BioRad | Cat# 1610373 |
Additional files
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Supplementary file 1
Plasmids used in the study and individual sgRNA sequences used.
Plasmids_Used: plasmid table for this study. sgRNAs_Used: sgRNA sequences used for individual sgRNA sequences.
- https://cdn.elifesciences.org/articles/69142/elife-69142-supp1-v2.xlsx
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Supplementary file 2
Information about sgRNA libraries related to Figure 2—figure supplement 1 and to Figure 3.
Sequencing and CasTLE analysis results from the whole-genome screen and sublibrary screens. Comparison of individual validation data to sublibrary screen data. WGS_sgRNAs: sgRNA sequences and target genes in the whole-genome screen. TA_WGS: CasTLE analysis of the whole-genome screen (TA screen HiLITR configuration). Sublibrary_sgRNAs: sgRNA sequences and target genes in the sublibrary screens. TA/SA/ER_Sublibrary: CasTLE analysis of the sublibrary screens (TA/SA/ER screen HiLITR configurations). Sublibrary_Comparison: comparison of combined-replicate CasTLE analysis across the TA/SA/ER sublibrary screens. Hits&Validation: sublibrary screen data for genes mentioned in main and supplementary figures, with independent validation data appended where applicable.
- https://cdn.elifesciences.org/articles/69142/elife-69142-supp2-v2.xlsx
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Supplementary file 3
Data from the proteomic analysis related to Figure 5 and Figure 5—figure supplements 3–5.
Results_MedNormed: abundance values and statistical analysis of experimental replicates, normalized to median abundance value in the replicate/column. Results_MitoNormed: abundance values and statistical analysis of experimental replicates, normalized to mean abundance value across mitochondrial proteins in the replicate/column. Results_ER-Normed: abundance values and statistical analysis of experimental replicates, normalized to mean abundance value across ER proteins in the replicate/column.
- https://cdn.elifesciences.org/articles/69142/elife-69142-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69142/elife-69142-transrepform1-v2.docx
