Home LiteratureArticle Details
PMID: 30581143 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation.

Molecular cell ·Vol. 73 ·No. 2 ·2019-00-17 ·Pages 377-389.e11

Leto DE, Morgens DW, Zhang L, Walczak CP, Elias JE, Bassik MC, Kopito RR

Abstract

The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.

MeSH Terms
CRISPR-Associated Protein 9/genetics,metabolism CRISPR-Cas Systems Clustered Regularly Interspaced Short Palindromic Repeats Endoplasmic Reticulum-Associated Degradation/drug effects,genetics Genome-Wide Association Study/methods HEK293 Cells Humans K562 Cells Kinetics Proteasome Endopeptidase Complex/genetics,metabolism Protein Folding Proteolysis Proteostasis/drug effects,genetics Ricin/pharmacology Substrate Specificity Ubiquitin-Protein Ligases/genetics,metabolism Ubiquitination
Chemicals
Ricin SYVN1 protein, human Ubiquitin-Protein Ligases CRISPR-Associated Protein 9 Proteasome Endopeptidase Complex
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Leto Dara E
Department of Biology, Stanford University, Stanford, CA 94305, USA.
Morgens David W
Department of Genetics, Stanford University, Stanford, CA 94305, USA.
Zhang Lichao
Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA.
Walczak Christopher P
Department of Biology, Stanford University, Stanford, CA 94305, USA.
Elias Joshua E
Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA.
Bassik Michael C
Department of Genetics, Stanford University, Stanford, CA 94305, USA; Program in Chemistry, Engineering and Medicine for Human Health (ChEM-H), Stanford University, Stanford, CA 94305, USA.
Kopito Ron R
Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address: kopito@stanford.edu.
References (61)
61 references, click to expand
  1. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.
    J Am Soc Mass Spectrom. 1994 Nov;5(11):976-89 PMID: 24226387
  2. Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins.
    J Cell Biol. 2014 Jun 23;205(6):847-62 PMID: 24958774
  3. Degradation signal masking by heterodimerization of MATalpha2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway.
    Cell. 1998 Jul 24;94(2):217-27 PMID: 9695950
  4. K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of k29 polyubiquitin.
    Mol Cell. 2015 Apr 2;58(1):83-94 PMID: 25752573
  5. Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.
    EMBO J. 1998 May 15;17(10):2759-66 PMID: 9582269
  6. MARCH6 and TRC8 facilitate the quality control of cytosolic and tail-anchored proteins.
    EMBO Rep. 2018 May;19(5): PMID: 29519897
  7. K63 ubiquitylation triggers proteasomal degradation by seeding branched ubiquitin chains.
    Proc Natl Acad Sci U S A. 2018 Feb 13;115(7):E1401-E1408 PMID: 29378950
  8. Defining human ERAD networks through an integrative mapping strategy.
    Nat Cell Biol. 2011 Nov 27;14(1):93-105 PMID: 22119785
  9. KCMF1 (potassium channel modulatory factor 1) Links RAD6 to UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4) and lysosome-mediated degradation.
    Mol Cell Proteomics. 2015 Mar;14(3):674-85 PMID: 25582440
  10. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.
    Nat Methods. 2007 Mar;4(3):207-14 PMID: 17327847
  11. Role of HERP and a HERP-related protein in HRD1-dependent protein degradation at the endoplasmic reticulum.
    J Biol Chem. 2014 Feb 14;289(7):4444-54 PMID: 24366871
  12. Site-directed mutagenesis of ricin A-chain and implications for the mechanism of action.
    Proteins. 1991;10(3):270-8 PMID: 1881883
  13. Signal peptide peptidase functions in ERAD to cleave the unfolded protein response regulator XBP1u.
    EMBO J. 2014 Nov 3;33(21):2492-506 PMID: 25239945
  14. Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.
    Cell. 2006 Jul 28;126(2):361-73 PMID: 16873066
  15. Sterol-regulated degradation of Insig-1 mediated by the membrane-bound ubiquitin ligase gp78.
    J Biol Chem. 2006 Dec 22;281(51):39308-15 PMID: 17043353
  16. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.
    Genome Biol. 2009;10(3):R25 PMID: 19261174
  17. Translocation of TCR alpha chains into the lumen of the endoplasmic reticulum and their degradation.
    Science. 1993 Mar 26;259(5103):1901-4 PMID: 8456316
  18. On the road to nowhere: cross-talk between post-translational protein targeting and cytosolic quality control.
    Biochem Soc Trans. 2016 Jun 15;44(3):796-801 PMID: 27284044
  19. The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology.
    Physiol Rev. 2012 Apr;92(2):537-76 PMID: 22535891
  20. The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates.
    J Biol Chem. 2013 Nov 29;288(48):34575-87 PMID: 24158444
  21. SEL1L-dependent Substrates Require Derlin2/3 and Herp1/2 for Endoplasmic Reticulum-associated Degradation.
    Cell Struct Funct. 2017 Jul 4;42(2):81-94 PMID: 28552883
  22. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens.
    Nat Commun. 2017 May 05;8:15178 PMID: 28474669
  23. A third-generation lentivirus vector with a conditional packaging system.
    J Virol. 1998 Nov;72(11):8463-71 PMID: 9765382
  24. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry.
    Nat Commun. 2014 Aug 27;5:4763 PMID: 25159004
  25. The ubiquitin code.
    Annu Rev Biochem. 2012;81:203-29 PMID: 22524316
  26. Global analysis of protein expression in yeast.
    Nature. 2003 Oct 16;425(6959):737-41 PMID: 14562106
  27. A HECT domain E3 enzyme assembles novel polyubiquitin chains.
    J Biol Chem. 2001 Jun 8;276(23):19871-8 PMID: 11278995
  28. How ricin and Shiga toxin reach the cytosol of target cells: retrotranslocation from the endoplasmic reticulum.
    Curr Top Microbiol Immunol. 2012;357:19-40 PMID: 21761287
  29. Redundant and Antagonistic Roles of XTP3B and OS9 in Decoding Glycan and Non-glycan Degrons in ER-Associated Degradation.
    Mol Cell. 2018 May 3;70(3):516-530.e6 PMID: 29706535
  30. Unassembled CD147 is an endogenous endoplasmic reticulum-associated degradation substrate.
    Mol Biol Cell. 2012 Dec;23(24):4668-78 PMID: 23097496
  31. Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification.
    Nat Chem Biol. 2016 May;12(5):361-6 PMID: 27018887
  32. Impairment of the ubiquitin-proteasome system by protein aggregation.
    Science. 2001 May 25;292(5521):1552-5 PMID: 11375494
  33. Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP.
    J Cell Biol. 1996 Feb;132(3):291-8 PMID: 8636208
  34. Ricin trafficking in cells.
    Toxins (Basel). 2015 Jan 09;7(1):49-65 PMID: 25584427
  35. Dislocation of ricin toxin A chains in human cells utilizes selective cellular factors.
    J Biol Chem. 2011 Jun 17;286(24):21231-8 PMID: 21527639
  36. Using linkage-specific monoclonal antibodies to analyze cellular ubiquitylation.
    Methods Mol Biol. 2012;832:185-96 PMID: 22350886
  37. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method.
    BMC Biotechnol. 2011 Oct 12;11:92 PMID: 21992524
  38. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
    Nat Protoc. 2007;2(8):1896-906 PMID: 17703201
  39. An internal polyadenylation signal substantially increases expression levels of lentivirus-delivered transgenes but has the potential to reduce viral titer in a promoter-dependent manner.
    Hum Gene Ther. 2008 Aug;19(8):840-50 PMID: 18627247
  40. Assembly and specific recognition of k29- and k33-linked polyubiquitin.
    Mol Cell. 2015 Apr 2;58(1):95-109 PMID: 25752577
  41. Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis.
    Mol Cell. 2014 Nov 6;56(3):360-75 PMID: 25284222
  42. A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks.
    Cell. 2015 Jul 30;162(3):675-86 PMID: 26189680
  43. A tissue-specific atlas of mouse protein phosphorylation and expression.
    Cell. 2010 Dec 23;143(7):1174-89 PMID: 21183079
  44. Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.
    Cell. 2017 Nov 2;171(4):918-933.e20 PMID: 29033132
  45. OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD.
    Nat Cell Biol. 2008 Mar;10(3):272-82 PMID: 18264092
  46. Systematic identification of interactions between host cell proteins and E7 oncoproteins from diverse human papillomaviruses.
    Proc Natl Acad Sci U S A. 2012 Jan 31;109(5):E260-7 PMID: 22232672
  47. Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities.
    Cell. 2006 Dec 29;127(7):1401-13 PMID: 17190603
  48. Folding-competent and folding-defective forms of ricin A chain have different fates after retrotranslocation from the endoplasmic reticulum.
    Mol Biol Cell. 2010 Aug 1;21(15):2543-54 PMID: 20519439
  49. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes.
    Nat Biotechnol. 2016 Jun;34(6):634-6 PMID: 27159373
  50. Quality control of glycoprotein folding and ERAD: the role of N-glycan handling, EDEM1 and OS-9.
    Histochem Cell Biol. 2017 Feb;147(2):269-284 PMID: 27803995
  51. The mammalian endoplasmic reticulum-associated degradation system.
    Cold Spring Harb Perspect Biol. 2013 Sep 01;5(9): PMID: 23232094
  52. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death.
    Nat Chem Biol. 2013 Sep;9(9):548-56 PMID: 23892893
  53. The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Membrane Proteins.
    Mol Cell. 2018 Mar 1;69(5):915 PMID: 29499140
  54. Ubiquitin-specific protease 2 as a tool for quantification of total ubiquitin levels in biological specimens.
    Anal Biochem. 2006 Jun 1;353(1):153-5 PMID: 16643835
  55. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions.
    Nat Biotechnol. 2017 May;35(5):463-474 PMID: 28319085
  56. Cleaning up in the endoplasmic reticulum: ubiquitin in charge.
    Nat Struct Mol Biol. 2014 Apr;21(4):325-35 PMID: 24699081
  57. The increasing complexity of the ubiquitin code.
    Nat Cell Biol. 2016 May 27;18(6):579-86 PMID: 27230526
  58. Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogues.
    J Biol Chem. 2011 Nov 25;286(47):40867-77 PMID: 21969368
  59. Enhanced protein degradation by branched ubiquitin chains.
    Cell. 2014 May 8;157(4):910-21 PMID: 24813613
  60. Membrane Protein Quantity Control at the Endoplasmic Reticulum.
    J Membr Biol. 2017 Aug;250(4):379-392 PMID: 27743014
  61. Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control.
    J Cell Biol. 2004 Apr;165(1):41-52 PMID: 15078901
Article Info
Journal
Molecular cell
Abbr.
Mol Cell
ISSN
1097-4164
DOI
10.1016/j.molcel.2018.11.015
Email
kopito@stanford.edu
Published
2019-00-17
Epub
2018-00-20
Pages
377-389.e11
Language
English
Region
United States
NLM ID
9802571
PMCID
PMC6338494
Subset
IM
Grants
NIGMS NIH HHS · F32 GM113370 · United States
NIGMS NIH HHS · F32 GM113378 · United States
NCRR NIH HHS · S10 RR027431 · United States
NIGMS NIH HHS · R01 GM074874 · United States
NCRR NIH HHS · S10 RR025518 · United States
External Links
PubMed source DOI: 10.1016/j.molcel.2018.11.015 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2