Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. doi: 10.1039/c1mb05175j.Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. Secretory pathway genes assessed by high-throughput microscopy and synthetic genetic array analysis. doi: 10.7554/eLife.21470.īircham PW, Maass DR, Roberts CA, Kiew PY, Low YS, Yegambaram M, Matthews J, Jack CA, Atkinson PH. EMC1-dependent stabilization drives membrane penetration of a partially destabilized non-enveloped virus. The SND proteins constitute an alternative targeting route to the endoplasmic reticulum. doi: 10.1016/j.celrep.2017.12.006.Īviram N, Ast T, Costa EA, Arakel EC, Chuartzman SG, Jan CH, Haßdenteufel S, Dudek J, Jung M, Schorr S, Zimmermann R, Schwappach B, Weissman JS, Schuldiner M. Identification of Oxa1 homologs operating in the eukaryotic endoplasmic reticulum. Development and characterization of a reconstituted yeast translation initiation system. Inset: Protein classifications pie chart for EMC client proteins (N = 37).Īlgire MA, Maag D, Savio P, Acker MG, Tarun SZ, Sachs AB, Asano K, Nielsen KH, Olsen DS, Phan L, Hinnebusch AG, Lorsch JR. ( F) Top non-redundant over-represented GOMF terms calculated from PANTHER (FDR < 0.05 redundant terms removed by REViGO). Blue boxes indicate 95% confidence ranges defined by 10,000 random sub-samplings of total TMDs with a pool size equal to EMC TMDs (N = 37). Proportion of EMC TMD amino acids/all TMDs for each property is shown by a blue line. ( E) Fraction of amino acids with the given properties in TMDs from EMC client proteins compared to all secretory proteins. ( D) Histogram showing the proportion of proteins containing the given number of TMDs for all proteins that enter the secretory pathway (as defined in Uniprot) and EMC clients. ( C) Comparison of translation change by ribosome profiling with proteome change. Type I membrane proteins have a cleavable ER-signal sequence, a single transmembrane. There are three types of single-pass transmembrane proteins with a single domain traversing through the membrane. Proteins colored green are significantly downregulated in both EMC2 and EMC4 cells (Log2 >0.5). Such proteins are inserted into the ER membrane co-translationally and are broadly categorized as single-pass and multipass transmembrane proteins. Proteins colored red are significantly upregulated in both EMC2 and EMC4 cells. Expression is relative to non-targeting GAL4 guide RNA. Multipass transmembrane protein full#( B) Full proteome comparison scatter plot of protein abundance change in cells expressing EMC2 guide RNA against abundance change in cells expressing EMC4 guide. PARK7 is shown as a control (Wiśniewski and Mann, 2016). ( A) SILAC quantification of EMC components, comparing their expression level in cells with guide RNAs targeting EMC2 or EMC4 with those expressing non-targeting GAL4 guide RNA. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.ĮMC cell biology endoplasmic reticulum human ion channel transmembrane transporter. 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. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. 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. 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. The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity.
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