A distinct dimer configuration of a diatom Get3 forming a tetrameric complex with its tail-anchored membrane cargo.

BACKGROUND: Most tail-anchored (TA) membrane proteins are delivered to the endoplasmic reticulum through a conserved posttranslational pathway. Although core mechanisms underlying the targeting and insertion of TA proteins are well established in eukaryotes, their role in mediating TA protein biogenesis in plants remains unclear. We reported the crystal structures of algal arsenite transporter 1 (ArsA1), which possesses an approximately 80-kDa monomeric architecture and carries chloroplast-localized TA proteins. However, the mechanistic basis of ArsA2, a Get3 (guided entry of TA proteins 3) homolog in plants, for TA recognition remains unknown. RESULTS: Here, for the first time, we present the crystal structures of the diatom Pt-Get3a that forms a distinct ellipsoid-shaped tetramer in the open (nucleotide-bound) state through crystal packing. Pulldown assay results revealed that only tetrameric Pt-Get3a can bind to TA proteins. The lack of the conserved zinc-coordination CXXC motif in Pt-Get3a potentially leads to the spontaneous formation of a distinct parallelogram-shaped dimeric conformation in solution, suggesting a new dimer state for subsequent tetramerization upon TA targeting. Pt-Get3a nonspecifically binds to different subsets of TA substrates due to the lower hydrophobicity of its α-helical subdomain, which is implicated in TA recognition. CONCLUSIONS: Our study provides new insights into the mechanisms underlying TA protein shielding by tetrameric Get3 during targeting to the diatom's cell membrane.

Structural analysis of chloroplast tail-anchored membrane protein recognition by ArsA1.

In mammals and yeast, tail-anchored (TA) membrane proteins destined for the post-translational pathway are safely delivered to the endoplasmic reticulum (ER) membrane by a well-known targeting factor, TRC40/Get3. In contrast, the underlying mechanism for translocation of TA proteins in plants remains obscure. How this unique eukaryotic membrane-trafficking system correctly distinguishes different subsets of TA proteins destined for various organelles, including mitochondria, chloroplasts and the ER, is a key question of long standing. Here, we present crystal structures of algal ArsA1 (the Get3 homolog) in a distinct nucleotide-free open state and bound to adenylyl-imidodiphosphate. This approximately 80-kDa protein possesses a monomeric architecture, with two ATPase domains in a single polypeptide chain. It is capable of binding chloroplast (TOC34 and TOC159) and mitochondrial (TOM7) TA proteins based on features of its transmembrane domain as well as the regions immediately before and after the transmembrane domain. Several helices located above the TA-binding groove comprise the interlocking hook-like motif implicated by mutational analyses in TA substrate recognition. Our data provide insights into the molecular basis of the highly specific selectivity of interactions of algal ArsA1 with the correct sets of TA substrates before membrane targeting in plant cells.

Epinecidin-1 protects mice from LPS-induced endotoxemia and cecal ligation and puncture-induced polymicrobial sepsis.

The antimicrobial peptide, epinecidin-1 (Epi), was identified from Epinephelus coioides and may have clinical application for treating sepsis. Epi has been shown to ameliorate antibiotic-resistant bacteria-induced sepsis in mice, but further evaluation in mixed-flora models and a description of the protective mechanisms are essential to establish this peptide as a potential therapeutic. Therefore, we first tested the protective effects of Epi against polymicrobial sepsis-induced bactericidal infection, inflammation and lung injury that result from cecal ligation and puncture in mice. Furthermore, since lipopolysaccharide (LPS) is a key inducer of inflammation during bacterial infection and sepsis, we also tested the LPS-antagonizing activity and related mechanisms of Epi-mediated protection in mice with LPS-induced endotoxemia and LPS-treated Raw264.7 mouse macrophage cells. Epi rescued mice from both polymicrobial sepsis and endotoxemia after delayed administration and suppressed both lung and systemic inflammatory responses, while attenuating lung injury and diminishing bacterial load. In vitro studies revealed that Epi suppressed LPS-induced inflammatory cytokine production. Mechanistically, Epi disrupted the interaction between LPS and LPS binding protein, competed with LPS for binding on the cell surface, and inhibited Toll-like receptor 4 endocytosis, resulting in inhibition of LPS-induced reactive oxygen species/p38/Akt/NF-κB signaling and subsequent cytokine production. Overall, our results demonstrate that Epi is a promising therapeutic agent for endotoxemia and polymicrobial sepsis.

Structural and functional characterization of ybr137wp implicates its involvement in the targeting of tail-anchored proteins to membranes.

Nearly 5% of membrane proteins are guided to nuclear, endoplasmic reticulum (ER), mitochondrial, Golgi, or peroxisome membranes by their C-terminal transmembrane domain and are classified as tail-anchored (TA) membrane proteins. In Saccharomyces cerevisiae, the guided entry of TA protein (GET) pathway has been shown to function in the delivery of TA proteins to the ER. The sorting complex for this pathway is comprised of Sgt2, Get4, and Get5 and facilitates the loading of nascent tail-anchored proteins onto the Get3 ATPase. Multiple pulldown assays also indicated that Ybr137wp associates with this complex in vivo. Here, we report a 2.8-Å-resolution crystal structure for Ybr137wp from Saccharomyces cerevisiae. The protein is a decamer in the crystal and also in solution, as observed by size exclusion chromatography and analytical ultracentrifugation. In addition, isothermal titration calorimetry indicated that the C-terminal acidic motif of Ybr137wp interacts with the tetratricopeptide repeat (TPR) domain of Sgt2. Moreover, an in vivo study demonstrated that Ybr137wp is induced in yeast exiting the log phase and ameliorates the defect of TA protein delivery and cell viability derived by the impaired GET system under starvation conditions. Therefore, this study suggests a possible role for Ybr137wp related to targeting of tail-anchored proteins.

Structure of the Sgt2 dimerization domain complexed with the Get5 UBL domain involved in the targeting of tail-anchored membrane proteins to the endoplasmic reticulum.

The insertion of tail-anchored membrane (TA) proteins into the appropriate membrane is a post-translational event that requires stabilization of the transmembrane domain and targeting to the proper destination. Sgt2, a small glutamine-rich tetratricopeptide-repeat protein, is a heat-shock protein cognate (HSC) co-chaperone that preferentially binds endoplasmic reticulum-destined TA proteins and directs them to the GET pathway via Get4 and Get5. The N-terminal domain of Sgt2 seems to exert dual functions. It mediates Get5 interaction and allows substrate delivery to Get3. Following the N-terminus of Get5 is a ubiquitin-like (Ubl) domain that interacts with the N-terminus of Sgt2. Here, the crystal structure of the Sgt2 dimerization domain complexed with the Get5 Ubl domain (Sgt2N-Get5Ubl) is reported. This complex reveals an intimate interaction between one Sgt2 dimer and one Get5 monomer. This research further demonstrates that hydrophobic residues from both Sgt2 and Get5 play an important role in cell survival under heat stress. This study provides detailed molecular insights into the specific binding of this GET-pathway complex.

Interaction surface and topology of Get3-Get4-Get5 protein complex, involved in targeting tail-anchored proteins to endoplasmic reticulum.

Recent work has uncovered the "GET system," which is responsible for endoplasmic reticulum targeting of tail-anchored proteins. Although structural information and the individual roles of most components of this system have been defined, the interactions and interplay between them remain to be elucidated. Here, we investigated the interactions between Get3 and the Get4-Get5 complex from Saccharomyces cerevisiae. We show that Get3 interacts with Get4-Get5 via an interface dominated by electrostatic forces. Using isothermal titration calorimetry and small-angle x-ray scattering, we further demonstrate that the Get3 homodimer interacts with two copies of the Get4-Get5 complex to form an extended conformation in solution.