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      The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins

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          Abstract

          The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.

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          Most cited references 54

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          Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
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            Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.

            Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale. Copyright © 2013 Elsevier Inc. All rights reserved.
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              CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.

              The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells. Copyright © 2013 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                29 May 2018
                2018
                : 7
                Affiliations
                [1 ]deptDepartment of Cellular and Molecular Pharmacology University of California, San Francisco San FranciscoUnited States
                [2 ]deptDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry MartinsriedGermany
                [3 ]deptDepartment of Biochemistry and Biophysics University of California, San Francisco San FranciscoUnited States
                [4 ]deptDepartment of Biological Sciences University of Pittsburgh PittsburghUnited States
                [5 ]deptDepartment of Pharmaceutical Chemistry University of California, San Francisco San FranciscoUnited States
                [6 ]Chan Zuckerberg Biohub San FranciscoUnited States
                [7 ]Howard Hughes Medical Institute, University of California, San Francisco San FranciscoUnited States
                University of Cambridge United Kingdom
                University of Cambridge United Kingdom
                Author notes
                [‡]

                Department of Molecular Biology, Princeton University, Princeton, United States.

                [§]

                Calico Life Sciences LLC, San Francisco, United States.

                [†]

                These authors contributed equally to this work.

                37018
                10.7554/eLife.37018
                5995541
                29809151
                © 2018, Shurtleff et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                Product
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
                Award ID: Investigator Program
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: GM075061
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100005237, Helen Hay Whitney Foundation;
                Award ID: Postdoctoral Fellowship
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100001033, Jane Coffin Childs Memorial Fund for Medical Research;
                Award ID: Postdoctoral Fellowship
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100007100, Sandler Foundation;
                Award ID: Program for Breakthrough Biomedical Research
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100008925, American Asthma Foundation;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001706, Louis-Jeantet Foundation;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100005984, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004189, Max-Planck-Gesellschaft;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award ID: Gottfried Wilhelm Leibniz Prize MA 1764/2-1
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100000781, European Research Council;
                Award ID: ERC2012-SyG_318987-ToPAG
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
                Award ID: Faculty Scholar Grant
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: AG041826
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: 1DP2GM110772-01
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: 8P41GM103481
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: 1S10OD16229
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Cell Biology
                Custom metadata
                The ER membrane protein complex promotes the biogenesis of a subset multipass membrane proteins enriched for transporters and other proteins with destabilizing features in transmembrane domains.

                Life sciences

                human, ion channel, transporter, transmembrane, endoplasmic reticulum, emc

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