Systematic simulation of the interactions of Pleckstrin homology domains with membranes

Pleckstrin homology (PH) domains can recruit proteins to membranes by recognition of phosphatidylinositol phosphates (PIPs). Here we report the systematic simulation of the interactions of 100 mammalian PH domains with PIP containing model membranes. Comparison with crystal structures of PH domains bound to PIP analogues demonstrates that our method correctly identifies interactions at known canonical and non-canonical sites, while revealing additional functionally important sites for interaction not observed in the crystal structure, such as for P-Rex1 and Akt1. At the family level, we find that the β1 and β2 strands and their connecting loop constitute the primary PIP interaction site for the majority of PH domains, but we highlight interesting exceptional cases. Simultaneous interaction with multiple PIPs and clustering of PIPs induced by PH domain binding are also observed. Our findings support a general paradigm for PH domain membrane association involving multivalent interactions with anionic lipids.

Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manipulates the host phagosome maturation pathway to replicate intracellularly. Mycobacterium marinum, a closely-related species, and Dictyostelium discoideum, a social amoeba and alternative phagocytic host, have been used as models to study host-pathogen interactions occurring during mycobacterial infections. Vacuolins, functional homologues of the mammalian flotillins, organize membrane microdomains and play a role in vesicular trafficking. Various pathogens have been reported to manipulate their membrane association and function. During infection of D. discoideum with M. marinum, Vacuolin C was specifically and highly induced and all three vacuolin isoforms were enriched at the mycobacteria-containing-vacuole (MCV). In addition, absence of vacuolins reduced escape from the MCV and conferred resistance to M. marinum infection. Moreover, ESAT-6, the membrane-disrupting virulence factor of M. marinum, was less associated with membranes when vacuolins were absent. Together, these results suggest that vacuolins are important host factors that are manipulated by mycobacteria to inflict membrane damage and escape from their compartment.

Light-triggered and phosphorylation-dependent 14-3-3 association with NON-PHOTOTROPIC HYPOCOTYL 3 is required for hypocotyl phototropism

NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of the auxin-dependent plant phototropic growth response. We report that NPH3 directly binds polyacidic phospholipids, required for plasma membrane association in darkness. We further demonstrate that blue light induces an immediate phosphorylation of a C-terminal 14-3-3 binding motif in NPH3. Subsequent association of 14-3-3 proteins is causal for the light-induced release of NPH3 from the membrane and accompanied by NPH3 dephosphorylation. In the cytosol, NPH3 dynamically transitions into membraneless condensate-like structures. The dephosphorylated state of the 14-3-3 binding site and NPH3 membrane recruitment are recoverable in darkness. NPH3 variants that constitutively localize either to the membrane or to condensates are non-functional, revealing a fundamental role of the 14-3-3 mediated dynamic change in NPH3 localization for auxin-dependent phototropism. This regulatory mechanism might be of general nature, given that several members of the NPH3-like family interact with 14-3-3 via a C-terminal motif.

PD-L1 degradation is regulated by electrostatic membrane association of its cytoplasmic domain

The cytoplasmic domain of PD-L1 (PD-L1-CD) regulates PD-L1 degradation and stability through various mechanism, making it an attractive target for blocking PD-L1-related cancer signaling. Here, by using NMR and biochemical techniques we find that the membrane association of PD-L1-CD is mediated by electrostatic interactions between acidic phospholipids and basic residues in the N-terminal region. The absence of the acidic phospholipids and replacement of the basic residues with acidic residues abolish the membrane association. Moreover, the basic-to-acidic mutations also decrease the cellular abundance of PD-L1, implicating that the electrostatic interaction with the plasma membrane mediates the cellular levels of PD-L1. Interestingly, distinct from its reported function as an activator of AMPK in tumor cells, the type 2 diabetes drug metformin enhances the membrane dissociation of PD-L1-CD by disrupting the electrostatic interaction, thereby decreasing the cellular abundance of PD-L1. Collectively, our study reveals an unusual regulatory mechanism that controls the PD-L1 level in tumor cells, suggesting an alternative strategy to improve the efficacy of PD-L1-related immunotherapies.

Loose Morphology and High Dynamism of OSER Structures Induced by the Membrane Domain of HMG-CoA Reductase

The membrane domain of eukaryotic HMG-CoA reductase (HMGR) has the conserved capacity to induce endoplasmic reticulum (ER) proliferation and membrane association into Organized Smooth Endoplasmic Reticulum (OSER) structures. These formations develop in response to overexpression of particular proteins, but also occur naturally in cells of the three eukaryotic kingdoms. Here, we characterize OSER structures induced by the membrane domain of Arabidopsis HMGR (1S domain). Immunochemical confocal and electron microscopy studies demonstrate that the 1S:GFP chimera co-localizes with high levels of endogenous HMGR in several ER compartments, such as the ER network, the nuclear envelope, the outer and internal membranes of HMGR vesicles and the OSER structures, which we name ER-HMGR domains. After high-pressure freezing, ER-HMGR domains show typical crystalloid, whorled and lamellar ultrastructural patterns, but with wide heterogeneous luminal spaces, indicating that the native OSER is looser and more flexible than previously reported. The formation of ER-HMGR domains is reversible. OSER structures grow by incorporation of ER membranes on their periphery and progressive compaction to the inside. The ER-HMGR domains are highly dynamic in their formation versus their disassembly, their variable spherical-ovoid shape, their fluctuating borders and their rapid intracellular movement, indicating that they are not mere ER membrane aggregates, but active components of the eukaryotic cell.

Identification of a proline-kinked amphipathic α-helix downstream from the methyltransferase domain of a potexvirus replicase and its role in virus replication and perinuclear complex formation

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus , the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. NMR analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a GFP-fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA co-located during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions and in vitro high molecular weight complexes were formed by both replication-competent and -incompetent viral replicons, and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales . Substitution of the proline residue but not the other residues necessary for replication allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that a perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

Genomic remnants of ancestral hydrogen and methane metabolism in Archaea drive anaerobic carbon cycling

Methane metabolism is among the hallmarks of Archaea, originating very early in their evolution. Other than its two main complexes, methyl-CoM reductase (Mcr) and tetrahydromethanopterin-CoM methyltransferase (Mtr), there exist other genes called "methanogenesis markers" that are believed to participate in methane metabolism. Many of them are Domains of Unknown Function. Here we show that these markers emerged together with methanogenesis. Even if Mcr is lost, the markers and Mtr can persist resulting in intermediate metabolic states related to the Wood-Ljungdahl pathway. Beyond the markers, the methanogenic ancestor was hydrogenotrophic, employing the anaplerotic hydrogenases Eha and Ehb. The selective pressures acting on Eha, Ehb, and Mtr partially depend on their subunits' membrane association. Integrating the evolution of all these components, we propose that the ancestor of all methane metabolizers was an autotrophic H2/CO2 methanogen that could perhaps use methanol but not oxidize alkanes. Hydrogen-dependent methylotrophic methanogenesis has since emerged multiple times independently, both alongside a vertically inherited Mcr or from a patchwork of ancient transfers. Through their methanogenesis genomic remnants, Thorarchaeota and two newly reconstructed order-level lineages in Archaeoglobi and Bathyarchaeota act as metabolically versatile players in carbon cycling of anoxic environments across the globe.