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      Cryo-EM structure of the lysosomal chloride-proton exchanger CLC-7 in complex with OSTM1

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          Abstract

          The chloride-proton exchanger CLC-7 plays critical roles in lysosomal homeostasis and bone regeneration and its mutation can lead to osteopetrosis, lysosomal storage disease and neurological disorders. In lysosomes and the ruffled border of osteoclasts, CLC-7 requires a β-subunit, OSTM1, for stability and activity. Here, we present electron cryomicroscopy structures of CLC-7 in occluded states by itself and in complex with OSTM1, determined at resolutions up to 2.8 Å. In the complex, the luminal surface of CLC-7 is entirely covered by a dimer of the heavily glycosylated and disulfide-bonded OSTM1, which serves to protect CLC-7 from the degradative environment of the lysosomal lumen. OSTM1 binding does not induce large-scale rearrangements of CLC-7, but does have minor effects on the conformation of the ion-conduction pathway, potentially contributing to its regulatory role. These studies provide insights into the role of OSTM1 and serve as a foundation for understanding the mechanisms of CLC-7 regulation.

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          Inside the cells of mammals, acidic compartments called lysosomes are responsible for breaking down large molecules and worn-out cells parts so their components can be used again. Similar to lysosomes, specialized cells called osteoclasts require an acidic environment to degrade tissues in the bone. Both osteoclasts and lysosomes rely on a two-component protein complex to help them digest molecules. Mutations in the genes for both proteins are directly linked to human diseases including neurodegeneration and osteopetrosis – a disease characterized by dense and brittle bones.

          For the main protein in this complex, called CLC-7, to remain stable and perform its roles, it requires an accessory subunit known as OSTM1. CLC-7 is a transporter that funnels electrically charged particles into and out of the lysosome, which helps to maintain the environment inside the lysosome compartment. However, due to the tight partnership between CLC-7 and OTSM1, how they influence each other is poorly understood.

          To determine the roles of CLC-7 and OSTM1, Schrecker et al. looked at the structure of the complex using a technique called single particle electron microscopy, which allows proteins to be visualized almost down to the individual atom. The analysis revealed that OSTM1 covers almost the entire inside surface of CLC-7, protecting it from the acidic environment inside the lysosome and contributing to its stability. When the two subunits are bound together, OSTM1 also slightly changes the structure of the pore formed by CLC-7, suggesting that OSTM1 may regulate CLC-7 activity.

          Schrecker et al. have laid the foundation for understanding more about the activity and regulation of CLC-7 and OSTM1 in lysosomes and osteoclasts. The structures described also help explain previous findings, including why OSTM1 is important for the stability of CLC-7.

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          Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Role: Senior Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                04 August 2020
                2020
                : 9
                : e59555
                Affiliations
                [1]Structural Biology Program, Memorial Sloan Kettering Cancer Center New YorkUnited States
                Stanford University School of Medicine United States
                The University of Texas at Austin United States
                Stanford University School of Medicine United States
                University of Zürich Switzerland
                Author information
                https://orcid.org/0000-0001-8542-6657
                https://orcid.org/0000-0003-0496-0669
                Article
                59555
                10.7554/eLife.59555
                7440919
                32749217
                bafcb228-c771-4cd3-8765-1f1b304949e4
                © 2020, Schrecker 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.

                History
                : 01 June 2020
                : 29 July 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100014185, Searle Scholars Program;
                Award Recipient :
                Funded by: Josie Robertson Investigators Program;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: accessory subunit
                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
                Structural Biology and Molecular Biophysics
                Custom metadata
                Single-particle cryo-EM structures of CLC-7 in the presence and absence of its β-subunit, OSTM1, reveal insights into transporter regulation.

                Life sciences
                chloride transport,proton transport,clc,human
                Life sciences
                chloride transport, proton transport, clc, human

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