Structural basis for membrane insertion by the human ER membrane protein complex

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved co- and posttranslational insertase at the ER. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 angstroms by cryo–electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer

A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

Polyclonal anti–immunoglobulin G (anti-IgG) secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in Escherichia coli and could thus make secondary antibody production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore coupling. We demonstrate their superior performance in Western blotting, in both peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and superresolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols, and allow multitarget localization with primary IgGs from the same species and of the same class

Structural basis for membrane insertion by the human ER membrane protein complex

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved co- and posttranslational insertase at the ER. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 angstroms by cryo–electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer

Regulated assembly of the ER membrane protein complex

The assembly of nascent proteins into multi-subunit complexes is tightly regulated to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble subunits for function. Here we show that the kinase With no lysine 1 (WNK1), known for its role in hypertension and neuropathy, is required for assembly of the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and precludes binding of ubiquitin ligases, permitting assembly. Using biochemical reconstitution, we show that after EMC2 reaches the membrane, its interaction partners within the EMC displace WNK1, and similarly shield its exposed hydrophobic surfaces. This work describes an unexpected role for WNK1 in protein biogenesis, and defines the general requirements of an assembly factor that will apply across the proteome

WNK1 is an assembly factor for the human ER membrane protein complex

The assembly of nascent proteins into multi-subunit complexes is a tightly regulated process that must occur at high fidelity to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble cytosolic subunits to function. Here, we show that the kinase with no lysine 1 (WNK1), known for its role in hypertension and neuropathy, functions as an assembly factor for the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2–8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and directly competes for binding of E3 ubiquitin ligases, permitting assembly. Depletion of WNK1 thus destabilizes both the EMC and its membrane protein clients. This work describes an unexpected role for WNK1 in protein biogenesis and defines the general requirements of an assembly factor that will apply across the proteome

Xpo7 is a broad-spectrum exportin and a nuclear import receptor

Exportins bind cargo molecules in a RanGTP-dependent manner inside nuclei and transport them through nuclear pores to the cytoplasm. CRM1/Xpo1 is the best-characterized exportin because specific inhibitors such as leptomycin B allow straightforward cargo validations in vivo. The analysis of other exportins lagged far behind, foremost because no such inhibitors had been available for them. In this study, we explored the cargo spectrum of exportin 7/Xpo7 in depth and identified not only ∼200 potential export cargoes but also, surprisingly, ∼30 nuclear import substrates. Moreover, we developed anti-Xpo7 nanobodies that acutely block Xpo7 function when transfected into cultured cells. The inhibition is pathway specific, mislocalizes export cargoes of Xpo7 to the nucleus and import substrates to the cytoplasm, and allowed validation of numerous tested cargo candidates. This establishes Xpo7 as a broad-spectrum bidirectional transporter and paves the way for a much deeper analysis of exportin and importin function in the future

Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels.

International audienceGlycosaminoglycans (GAGs) are ubiquitously present at the cell surface and in extracellular matrix, and crucial for matrix assembly, cell-cell and cell-matrix interactions. The supramolecular presentation of GAG chains, along with other matrix components, is likely to be functionally important but remains challenging to control and to characterize, both in vivo and in vitro. We present a method to create well-defined biomimetic surfaces that display GAGs, either alone or together with other cell ligands, in a background that suppresses non-specific binding. Through the design of the immobilization platform - a streptavidin monolayer serves as a molecular breadboard for the attachment of various biotinylated ligands - and a set of surface-sensitive in situ analysis techniques (including quartz crystal microbalance and spectroscopic ellipsometry), the biomimetic surfaces are tailor made with tight control on biomolecular orientation, surface density and lateral mobility. Analysing the interactions between a selected GAG (heparan sulphate, HS) and the HS-binding chemokine CXCL12α (also called SDF-1α), we demonstrate that these surfaces are versatile for biomolecular and cellular interaction studies. T-lymphocytes are found to adhere specifically to surfaces presenting CXCL12α, both when reversibly bound through HS and when irreversibly immobilized on the inert surface, even in the absence of any bona fide cell adhesion ligand. Moreover, surfaces which present both HS-bound CXCL12α and the intercellular adhesion molecule 1 (ICAM-1) synergistically promote cell adhesion. Our surface biofunctionalization strategy should be broadly applicable for functional studies that require a well-defined supramolecular presentation of GAGs along with other matrix or cell-surface components

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope- mapping and native protein complex isolation

Abstract Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophoremaleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks -each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target