HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.

Stability of a biomembrane tube covered with proteins.

Membrane tubes are essential structural features in cells that facilitate biomaterial transport and inter- and intracellular signaling. The shape of these tubes can be regulated by the proteins that surround and adhere to them. We study the stability of a biomembrane tube coated with proteins by combining linear stability analysis, out-of-equilibrium hydrodynamic calculations, and numerical solutions of a Helfrich-like membrane model. Our analysis demonstrates that both long- and short-wavelength perturbations can destabilize the tubes. Numerical simulations confirm the derived linear stability criteria and yield the nonlinearly perturbed vesicle shapes. Our study highlights the interplay between membrane shape and protein density, where the shape instability concurs with a redistribution of proteins into a banded pattern.

Physiological and pathological functions of TMEM106B in neurodegenerative diseases.

As an integral lysosomal transmembrane protein, transmembrane protein 106B (TMEM106B) regulates several aspects of lysosomal function and is associated with neurodegenerative diseases. The TMEM106B gene mutations lead to lysosomal dysfunction and accelerate the pathological progression of Neurodegenerative diseases. Yet, the precise mechanism of TMEM106B in Neurodegenerative diseases remains unclear. Recently, different research teams discovered that TMEM106B is an amyloid protein and the C-terminal domain of TMEM106B forms amyloid fibrils in various Neurodegenerative diseases and normally elderly individuals. In this review, we discussed the physiological functions of TMEM106B. We also included TMEM106B gene mutations that cause neurodegenerative diseases. Finally, we summarized the identification and cryo-electronic microscopic structure of TMEM106B fibrils, and discussed the promising therapeutic strategies aimed at TMEM106B fibrils and the future directions for TMEM106B research in neurodegenerative diseases.

Protein Interface Regulating the Inserting Process of Imidazole Ionic Liquids into the Cell Membrane.

Ionic liquids (ILs) have shown promising potential in membrane protein extraction; however, the underlying mechanism remains unclear. Herein, we employed GPU-accelerated molecular dynamics (MD) simulations to investigate the dynamic insertion process of ILs into cell membranes containing membrane proteins. Our findings reveal that ILs spontaneously insert into the membrane, and the presence of membrane proteins significantly decelerates the rate of IL insertion into the membrane. Specifically, the relationship between the insertion rate and inserting free energy exhibits non-monotonic changes, which can be attributed to interfacial effects. The protein-water interface acts as trap for free ions and ionic clusters, while free ions preferentially insert into the membrane from the protein-lipid interface, which limits the insertion rate due to its narrowness. Thus, the insertion rate is governed by a combination of the free energy and interfacial effects. These findings provide valuable insights into the interfacial effects of protein-lipid bilayers and have implications for various biochemical-related applications.

Probing the energy barriers and stages of membrane protein unfolding using solid-state NMR spectroscopy.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

Getting membrane proteins into shape.

In this issue, Ji et al.1 show how a multipass membrane protein that initially inserts into the endoplasmic reticulum in a mostly inverted topology is post-translationally dislocated, re-inserted, and folded with the help of ATP13A1, a P-type ATPase.

An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

17O Solid-State NMR Spectroscopy of Lipid Membranes.

Despite the limitations posed by poor sensitivity, studies have reported the unique advantages of 17O based NMR spectroscopy to study systems existing in liquid, solid, or semisolid states. 17O NMR studies have exploited the remarkable sensitivity of quadrupole coupling and chemical shift anisotropy tensors to the local environment in the characterization of a variety of intra- and intermolecular interactions and motion. Recent studies have considerably expanded the use of 17O NMR to study dynamic intermolecular interactions associated with some of the challenging biological systems under magic angle spinning (MAS) and aligned conditions. The very fast relaxing nature of 17O has been well utilized in cellular and in vivo MRS (magnetic resonance spectroscopy) and MRI (magnetic resonance imaging) applications. The main focus of this Review is to highlight the new developments in the biological solids with a detailed discussion for a few selected examples including membrane proteins and nanodiscs. In addition to the unique benefits and limitations, the remaining challenges to overcome, and the impacts of higher magnetic fields and sensitivity enhancement techniques are discussed.

A new preparation method of covalent annular nanodiscs based on MTGase.

The preservation of the native conformation and functionality of membrane proteins has posed considerable challenges. While detergents and liposome reconstitution have been traditional approaches, nanodiscs (NDs) offer a promising solution by embedding membrane proteins in phospholipids encircled by an amphipathic helical protein MSP belt. Nevertheless, a drawback of commonly used NDs is their limited homogeneity and stability. In this study, we present a novel approach to construct covalent annular nanodiscs (cNDs) by leveraging microbial transglutaminase (MTGase) to catalyze isopeptide bond formation between the side chains of terminal amino acids, specifically Lysine (K) and Glutamine (Q). This methodology significantly enhances the homogeneity and stability of NDs. Characterization of cNDs and the assembly of membrane proteins within them validate the successful reconstitution of membrane proteins with improved homogeneity and stability. Our findings suggest that cNDs represent a more suitable tool for investigating interactions between membrane proteins and lipids, as well as for analyzing membrane protein structures.

Investigations of membrane protein interactions in cells using fluorescence microscopy.

The interactions between proteins in membranes govern many cellular functions. Our ability to probe for such interactions has greatly evolved in recent years due to the introduction of new fluorescence techniques. As a result, we currently have a choice of methods that can be used to assess the spatial distribution of a membrane protein, its association state, and the thermodynamic stability of the oligomers in the native milieu. These biophysical measurements have revealed new insights into important biological processes in cellular membranes.

Experimental and computational approaches for membrane protein insertion and topology determination.

Membrane proteins play pivotal roles in a wide array of cellular processes and constitute approximately a quarter of the protein-coding genes across all organisms. Despite their ubiquity and biological significance, our understanding of these proteins remains notably less comprehensive compared to their soluble counterparts. This disparity in knowledge can be attributed, in part, to the inherent challenges associated with employing specialized techniques for the investigation of membrane protein insertion and topology. This review will center on a discussion of molecular biology methodologies and computational prediction tools designed to elucidate the insertion and topology of helical membrane proteins.

The polarity protein Yurt associates with the plasma membrane via basic and hydrophobic motifs embedded in its FERM domain.

The subcellular distribution of the polarity protein Yurt (Yrt) is subjected to a spatio-temporal regulation in Drosophila melanogaster embryonic epithelia. After cellularization, Yrt binds to the lateral membrane of ectodermal cells and maintains this localization throughout embryogenesis. During terminal differentiation of the epidermis, Yrt accumulates at septate junctions and is also recruited to the apical domain. Although the mechanisms through which Yrt associates with septate junctions and the apical domain have been deciphered, how Yrt binds to the lateral membrane remains as an outstanding puzzle. Here, we show that the FERM domain of Yrt is necessary and sufficient for membrane localization. Our data also establish that the FERM domain of Yrt directly binds negatively charged phospholipids. Moreover, we demonstrate that positively charged amino acid motifs embedded within the FERM domain mediates Yrt membrane association. Finally, we provide evidence suggesting that Yrt membrane association is functionally important. Overall, our study highlights the molecular basis of how Yrt associates with the lateral membrane during the developmental time window where it is required for segregation of lateral and apical domains.

NMR methods to detect fluoride binding and transport by membrane proteins.

Solid-state nuclear magnetic resonance (NMR) methods can probe the motions of membrane proteins in liposomes at the atomic level, and propel the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. High-resolution crystallography snapshots have provided a structural basis for fluoride channels. NMR is a powerful tool to build upon these snapshots and depict a dynamic picture of fluoride channels in native-like lipid bilayers. In this contribution, we discuss solid-state and solution NMR experiments to detect fluoride binding and transport by fluoride channels. Ongoing developments in membrane protein sample preparation and ssNMR methodology, particularly in using 1H, 19F and 13C-detection schemes, offer additional opportunities to study structure and functional aspects of fluoride channels.

Electron videography of a lipid-protein tango.

Biological phenomena, from enzymatic catalysis to synaptic transmission, originate in the structural transformations of biomolecules and biomolecular assemblies in liquid water. However, directly imaging these nanoscopic dynamics without probes or labels has been a fundamental methodological challenge. Here, we developed an approach for "electron videography"-combining liquid phase electron microscopy with molecular modeling-with which we filmed the nanoscale structural fluctuations of individual, suspended, and unlabeled membrane protein nanodiscs in liquid. Systematic comparisons with biochemical data and simulation indicate the graphene encapsulation involved can afford sufficiently reduced effects of the illuminating electron beam for these observations to yield quantitative fingerprints of nanoscale lipid-protein interactions. Our results suggest that lipid-protein interactions delineate dynamically modified membrane domains across unexpectedly long ranges. Moreover, they contribute to the molecular mechanics of the nanodisc as a whole in a manner specific to the protein within. Overall, this work illustrates an experimental approach to film, quantify, and understand biomolecular dynamics at the nanometer scale.

Computational insights into the conformational transition of STING: Mechanistic, energetic considerations, and the influence of crucial mutations.

STING (stimulator of interferon genes) is a crucial protein in the innate immune system's response to viral and bacterial infections. In this study, we investigated the mechanistic and energetic mechanism of the conformational transition process of STING activated by cGAMP binding. We found that the STING connector region undergoes an energetically unfavorable rotation during this process, which is compensated by the favorable interaction between cGAMP and the STING ligand binding domain. We further studied several disease-causing mutations and found that the V155 M mutation facilitates a smoother transition in the STING connector region. However, the V147L mutation exhibits unfavorable conformational transition energy, suggesting it may hinder STING activation pathway that relies on connector region rotation. Despite being labeled as hyperactive, the widespread prevalence of V147L/V147I mutations across species implies a neutral character, indicating complexity in its role. Overall, our analysis deepens the understanding of STING activation within the connector region, and targeting this region with compounds may provide an alternative approach to interfering with STING's function.

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Structural insights into the molecular mechanism of phytoplasma immunodominant membrane protein.

Immunodominant membrane protein (IMP) is a prevalent membrane protein in phytoplasma and has been confirmed to be an F-actin-binding protein. However, the intricate molecular mechanisms that govern the function of IMP require further elucidation. In this study, the X-ray crystallographic structure of IMP was determined and insights into its interaction with plant actin are provided. A comparative analysis with other proteins demonstrates that IMP shares structural homology with talin rod domain-containing protein 1 (TLNRD1), which also functions as an F-actin-binding protein. Subsequent molecular-docking studies of IMP and F-actin reveal that they possess complementary surfaces, suggesting a stable interaction. The low potential energy and high confidence score of the IMP-F-actin binding model indicate stable binding. Additionally, by employing immunoprecipitation and mass spectrometry, it was discovered that IMP serves as an interaction partner for the phytoplasmal effector causing phyllody 1 (PHYL1). It was then shown that both IMP and PHYL1 are highly expressed in the S2 stage of peanut witches' broom phytoplasma-infected Catharanthus roseus. The association between IMP and PHYL1 is substantiated through in vivo immunoprecipitation, an in vitro cross-linking assay and molecular-docking analysis. Collectively, these findings expand the current understanding of IMP interactions and enhance the comprehension of the interaction of IMP with plant F-actin. They also unveil a novel interaction pathway that may influence phytoplasma pathogenicity and host plant responses related to PHYL1. This discovery could pave the way for the development of new strategies to overcome phytoplasma-related plant diseases.

NINJ1 mediates plasma membrane rupture by cutting and releasing membrane disks.

The membrane protein NINJ1 mediates plasma membrane rupture in pyroptosis and other lytic cell death pathways. Here, we report the cryo-EM structure of a NINJ1 oligomer segmented from NINJ1 rings. Each NINJ1 subunit comprises amphipathic (⍺1, ⍺2) and transmembrane (TM) helices (⍺3, ⍺4) and forms a chain of subunits, mainly by the TM helices and ⍺1. ⍺3 and ⍺4 are kinked, and the Gly residues are important for function. The NINJ1 oligomer possesses a concave hydrophobic side that should face the membrane and a convex hydrophilic side formed by ⍺1 and ⍺2, presumably upon activation. This structural observation suggests that NINJ1 can form membrane disks, consistent with membrane fragmentation by recombinant NINJ1. Live-cell and super-resolution imaging uncover ring-like structures on the plasma membrane that are released into the culture supernatant. Released NINJ1 encircles a membrane inside, as shown by lipid staining. Therefore, NINJ1-mediated membrane disk formation is different from gasdermin-mediated pore formation, resulting in membrane loss and plasma membrane rupture.

Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse.

A crucial event in sexual reproduction is when haploid sperm and egg fuse to form a new diploid organism at fertilization. In mammals, direct interaction between egg JUNO and sperm IZUMO1 mediates gamete membrane adhesion, yet their role in fusion remains enigmatic. We used AlphaFold to predict the structure of other extracellular proteins essential for fertilization to determine if they could form a complex that may mediate fusion. We first identified TMEM81, whose gene is expressed by mouse and human spermatids, as a protein having structural homologies with both IZUMO1 and another sperm molecule essential for gamete fusion, SPACA6. Using a set of proteins known to be important for fertilization and TMEM81, we then systematically searched for predicted binary interactions using an unguided approach and identified a pentameric complex involving sperm IZUMO1, SPACA6, TMEM81 and egg JUNO, CD9. This complex is structurally consistent with both the expected topology on opposing gamete membranes and the location of predicted N-glycans not modeled by AlphaFold-Multimer, suggesting that its components could organize into a synapse-like assembly at the point of fusion. Finally, the structural modeling approach described here could be more generally useful to gain insights into transient protein complexes difficult to detect experimentally.

Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are trapped in microfluidic devices for analysis by optical microscopy.