12
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          ABSTRACT

          The eukaryotic endoplasmic reticulum (ER) membrane contains essential complexes that oversee protein biogenesis and lipid metabolism, impacting nearly all aspects of cell physiology. The ER membrane protein complex (EMC) is a newly described transmembrane domain (TMD) insertase linked with various phenotypes, but whose clients and cellular responsibilities remain incompletely understood. We report that EMC deficiency limits the cellular boundaries defining cholesterol tolerance, reflected by diminished viability with limiting or excessive extracellular cholesterol. Lipidomic and proteomic analyses revealed defective biogenesis and concomitant loss of the TMD-containing ER-resident enzymes sterol-O-acyltransferase 1 (SOAT1) and squalene synthase (SQS, also known as FDFT1), which serve strategic roles in the adaptation of cells to changes in cholesterol availability. Insertion of the weakly hydrophobic tail-anchor (TA) of SQS into the ER membrane by the EMC ensures sufficient flux through the sterol biosynthetic pathway while biogenesis of polytopic SOAT1 promoted by the EMC provides cells with the ability to store free cholesterol as inert cholesteryl esters. By facilitating insertion of TMDs that permit essential mammalian sterol-regulating enzymes to mature accurately, the EMC is an important biogenic determinant of cellular robustness to fluctuations in cholesterol availability.

          This article has an associated First Person interview with the first author of the paper.

          Abstract

          [Related article:] Highlighted Article: The ER membrane protein complex promotes biogenesis of key membrane-bound enzymes responsible for regulation of cholesterol biosynthesis and storage, an important determinant of mammalian cell viability.

          Related collections

          Most cited references52

          • Record: found
          • Abstract: found
          • Article: found
          Is Open Access

          REAP: A two minute cell fractionation method

          Background The translocation or shuttling of proteins between the nucleus and cytoplasm (nucleocytoplasmic transport [NCPT]) is often a rapid event following stimulation with growth factors or in response to stress or other experimental manipulations. Commonly used methods to separate nuclei from cytoplasm employ lengthy steps such as density gradient centrifugation which exposes cells to non-physiological hyperosmotic conditions for extended time periods resulting in varying degrees of leakage between the nucleus and cytoplasm. To help maintain and quantify nuclear:cytoplasmic ratios of proteins, agents such as leptomycin B have been employed to be able to better analyze NCPT by inhibiting nuclear export. To track NCPT in the absence of these experimental manipulations that could introduce unknown artefacts, we have developed a rapid method that appears to produce pure nuclear and cytoplasmic fractions, suitable for obtaining accurate estimates of the nuclear:cytoplasmic ratios of proteins known to undergo NCPT. Findings We have developed a Rapid, Efficient And Practical (REAP) method for subcellular fractionation of primary and transformed human cells in culture. The REAP method is a two minute non-ionic detergent-based purification technique requiring only a table top centrifuge, micro-pipette and micro-centrifuge tubes. This inexpensive method has proven to efficiently separate nuclear from cytoplasmic proteins as estimated by no detectible cross-contamination of the nucleoporin and lamin A nuclear markers or the pyruvate kinase and tubulin cytoplasmic markers. REAP fractions also mirrored TNFα induced NF-κB NCPT observed in parallel by indirect immunofluorescence. Conclusions This method drastically reduces the time needed for subcellular fractionation, eliminates detectable protein degradation and maintains protein interactions. The simplicity, brevity and efficiency of this procedure allows for tracking ephemeral changes in subcellular relocalization of proteins while maintaining protein integrity and protein complex interactions.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Defining human ERAD networks through an integrative mapping strategy

            SUMMARY Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin and proteasome dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organised into a functional network that coordinates recognition, ubiquitination, and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organisation of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics, and the transcriptional response to ER stress. This analysis supports an adaptive organisation for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD.

              Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant alpha(1)-antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells.
                Bookmark

                Author and article information

                Journal
                J Cell Sci
                J. Cell. Sci
                JCS
                joces
                Journal of Cell Science
                The Company of Biologists Ltd
                0021-9533
                1477-9137
                15 January 2019
                16 January 2019
                16 January 2019
                : 132
                : 2
                : jcs223453
                Affiliations
                [1 ]Ludwig Institute for Cancer Research, University of Oxford, ORCRB , Headington, Oxford, OX3 7DQ, UK
                [2 ]Target Discovery Institute (TDI) Mass Spectrometry Laboratory , Nuffield Department of Medicine, University of Oxford , Headington, Oxford, OX3 7DQ, UK
                [3 ]Dept. of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology, University of California-Berkeley , Berkeley, CA, 94720, USA
                [4 ]MRC Laboratory of Molecular Biology , Francis Crick Avenue, Cambridge CB2 0QH, UK
                [5 ]Oxford Centre for Translational Myeloma Research, NDORMS, University of Oxford, Botnar Research Centre , Headington, Oxford, OX3 7LD, UK
                Author notes
                [*]

                Present address: Cambridge Institute for Medical Research, Hills Road, Cambridge Biomedical Campus, Cambridge, CB2 0XY, UK.

                []Author for correspondence ( john.christianson@ 123456ndorms.ox.ac.uk )
                Author information
                http://orcid.org/0000-0003-0766-5606
                http://orcid.org/0000-0002-3361-7264
                http://orcid.org/0000-0003-1614-8360
                http://orcid.org/0000-0001-8338-852X
                http://orcid.org/0000-0002-8160-2446
                http://orcid.org/0000-0002-0474-1207
                Article
                JCS223453
                10.1242/jcs.223453
                6362398
                30578317
                632f2c43-defb-4117-82bf-1d863b1bc362
                © 2019. Published by The Company of Biologists Ltd

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

                History
                : 7 August 2018
                : 3 December 2018
                Funding
                Funded by: Medical Research Council, http://dx.doi.org/10.13039/501100000265;
                Award ID: MR/L001209/1
                Funded by: Ludwig Institute for Cancer Research, http://dx.doi.org/10.13039/100009729;
                Award ID: MC_UP_A022_1007
                Funded by: National Institutes of Health, http://dx.doi.org/10.13039/100000002;
                Award ID: R01-CA172667
                Funded by: John Fell Fund, University of Oxford, http://dx.doi.org/10.13039/501100004789;
                Award ID: 133/075
                Funded by: Wellcome Trust, http://dx.doi.org/10.13039/100010269;
                Award ID: 097813/Z/11/Z
                Categories
                Research Article

                Cell biology
                emc,endoplasmic reticulum,squalene synthase,soat1,cholesterol homeostasis
                Cell biology
                emc, endoplasmic reticulum, squalene synthase, soat1, cholesterol homeostasis

                Comments

                Comment on this article