Membrane-associated Complex Research Articles

Immobilization of Membrane-Associated Protein Complexes on SERS-Active Nanomaterials for Structural and Dynamic Characterization.

Exploring the structural basis of membrane proteins is significant for a deeper understanding of protein functions. In situ analysis of membrane proteins and their dynamics, however, still challenges conventional techniques. Here we report the first attempt to immobilize membrane protein complexes on surface-enhanced Raman scattering (SERS)-active supports, titanium dioxide-coated silver (Ag@TiO2) nanoparticles. Biocompatible immobilization of microsomal monooxygenase complexes is achieved through lipid fission and fusion. SERS activity of the Ag@TiO2 nanoparticles enables in situ monitoring of protein-protein electron transfer and enzyme catalysis in real time. Through SERS fingerprints of the monooxygenase redox centers, the correlations between these protein-ligand interactions and reactive oxygen species generation are revealed, providing novel insights into the molecular mechanisms underlying monooxygenase-mediated apoptotic regulation. This study offers a novel strategy to explore structure-function relationships of membrane protein complexes and has the potential to advance the development of novel reactive oxygen species-inducing drugs for cancer therapy.

Conserved mechanism of Xrn1 regulation by glycolytic flux and protein aggregation

The regulation of gene expression in eukaryotes relies largely on the action of exoribonucleases, evolutionarily conserved enzymes that digest decapped messenger RNAs in the 5’-3’ direction. The activity of Xrn1, the major yeast exoribonuclease, is regulated by targeted changes in its cellular localisation in direct response to the cell’s metabolic state. When fermentable carbon sources are available, active Xrn1 is diffusely localised in the cytosol. Upon depletion of these sources, Xrn1 is sequestered at the plasma membrane-associated protein complex, the eisosome, and becomes inactive. Although this phenomenon has been described previously, the molecular mechanisms underlying these changes remain unknown. We report that the binding of Xrn1 to the plasma membrane is subject to glycolytic flux, rather than the availability of a fermentable carbon source, is independent of TORC1 activity and requires the core eisosomal proteins Pil1 and Lsp1. We identify the SH3-like domain of the Xrn1 protein as a putative interaction domain. In addition, we show that when expressed in Saccharomyces cerevisiae, the human orthologue of Xrn1 mirrors its yeast counterpart, i.e., it segregates to the eisosome under conditions of halted glycolysis. Our results not only advance our understanding of Xrn1 regulation but also indicate that this regulatory principle is conserved from yeast to humans.

Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1.

Nicotinamide adenine dinucleotide (NAD) is involved in renal physiology and is synthesized by nicotinamide mononucleotide adenylyltransferase (NMNAT). NMNAT exists as three isoforms, namely, NMNAT1, NMNAT2, and NMNAT3, encoded by Nmnat1, Nmnat2, and Nmnat3, respectively. In diabetic nephropathy (DN), NAD levels decrease, aggravating renal fibrosis. Conversely, sodium-glucose cotransporter-2 inhibitors increase NAD levels, mitigating renal fibrosis. In this regard, renal NAD synthesis has recently gained attention. However, the renal role of Nmnat in DN remains uncertain. Therefore, we investigated the role of Nmnat by establishing genetically engineered mice. Among the three isoforms, NMNAT1 levels were markedly reduced in the proximal tubules (PTs) of db/db mice. We examined the phenotypic changes in PT-specific Nmnat1 conditional knockout (CKO) mice. In CKO mice, Nmnat1 expression in PTs was downregulated when the tubules exhibited albuminuria, peritubular type IV collagen deposition, and mitochondrial ribosome (mitoribosome) excess. In CKO mice, Nmnat1 deficiency-induced mitoribosome excess hindered mitoribosomal translation of mitochondrial inner membrane-associated oxidative phosphorylation complex I (CI), CIII, CIV, and CV proteins and mitoribosomal dysfunction. Furthermore, the expression of hypermethylated in cancer 1, a transcription repressor, was downregulated in CKO mice, causing mitoribosome excess. Nmnat1 overexpression preserved mitoribosomal function, suggesting its protective role in DN.

The phage shock protein (PSP) envelope stress response: discovery of novel partners and evolutionary history.

Bacterial phage shock protein (PSP) systems stabilize the bacterial cell membrane and protect against envelope stress. These systems have been associated with virulence, but despite their critical roles, PSP components are not well characterized outside proteobacteria. Using comparative genomics and protein sequence-structure-function analyses, we systematically identified and analyzed PSP homologs, phyletic patterns, domain architectures, and gene neighborhoods. This approach underscored the evolutionary significance of the system, revealing that its core protein PspA (Snf7 in ESCRT outside bacteria) was present in the last universal common ancestor and that this ancestral functionality has since diversified into multiple novel, distinct PSP systems across life. Several novel partners of the PSP system were identified: (i) the Toastrack domain, likely facilitating assembly of sub-membrane stress-sensing and signaling complexes, (ii) the newly defined HTH-associated α-helical signaling domain-PadR-like transcriptional regulator pair system, and (iii) multiple independent associations with ATPase, CesT/Tir-like chaperone, and Band-7 domains in proteins thought to mediate sub-membrane dynamics. Our work also uncovered links between the PSP components and other domains, such as novel variants of SHOCT-like domains, suggesting roles in assembling membrane-associated complexes of proteins with disparate biochemical functions. Results are available at our interactive web app, https://jravilab.org/psp.IMPORTANCEPhage shock proteins (PSP) are virulence-associated, cell membrane stress-protective systems. They have mostly been characterized in Proteobacteria and Firmicutes. We now show that a minimal PSP system was present in the last universal common ancestor that evolved and diversified into newly identified functional contexts. Recognizing the conservation and evolution of PSP systems across bacterial phyla contributes to our understanding of stress response mechanisms in prokaryotes. Moreover, the newly discovered PSP modularity will likely prompt new studies of lineage-specific cell envelope structures, lifestyles, and adaptation mechanisms. Finally, our results validate the use of domain architecture and genetic context for discovery in comparative genomics.

Microcephaly with a disproportionate hippocampal reduction, stem cell loss and neuronal lipid droplet symptoms in Trappc9 KO mice

Mutations of the human TRAFFICKING PROTEIN PARTICLE COMPLEX SUBUNIT 9 (TRAPPC9) cause a neurodevelopmental disorder characterised by microcephaly and intellectual disability. Trappc9 constitutes a subunit specific to the intracellular membrane-associated TrappII complex. The TrappII complex interacts with Rab11 and Rab18, the latter being specifically associated with lipid droplets (LDs). Here we used non-invasive imaging to characterise Trappc9 knock-out (KO) mice as a model of the human hereditary disorder. KOs developed postnatal microcephaly with many grey and white matter regions being affected. In vivo magnetic resonance imaging (MRI) identified a disproportionately stronger volume reduction in the hippocampus, which was associated with a significant loss of Sox2-positive neural stem and progenitor cells. Diffusion tensor imaging indicated a reduced organisation or integrity of white matter areas. Trappc9 KOs displayed behavioural abnormalities in several tests related to exploration, learning and memory. Trappc9-deficient primary hippocampal neurons accumulated a larger LD volume per cell following Oleic Acid stimulation, and the coating of LDs by Perilipin-2 was much reduced. Additionally, Trappc9 KOs developed obesity, which was significantly more severe in females than in males. Our findings indicate that, beyond previously reported Rab11-related vesicle transport defects, dysfunctions in LD homeostasis might contribute to the neurobiological symptoms of Trappc9 deficiency.

The Bacillus subtilis cell envelope stress-inducible ytpAB operon modulates membrane properties and contributes to bacitracin resistance.

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σM responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σM-dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C35 terpenoids. Using targeted lipidomics, we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless, ytpA was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of ytpA increased membrane fluidity as monitored by fluorescence anisotropy. The ytpA gene contributes to bacitracin resistance in mutants additionally lacking the bceAB or bcrC genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which σM-dependent induction of the ytpAB operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes involved in cell wall homeostasis.IMPORTANCEPeptidoglycan synthesis inhibitors include some of our most important antibiotics. In Bacillus subtilis, peptidoglycan synthesis inhibitors induce the σM regulon, which is critical for intrinsic antibiotic resistance. The σM-dependent ytpAB operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C35 terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Furthermore, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the undecaprenyl-pyrophosphate lipid carrier that sustains peptidoglycan synthesis.

Disruption of the bacterial OLE RNP complex impairs growth on alternative carbon sources.

Ornate, large, extremophilic (OLE) RNAs comprise a class of large noncoding RNAs in bacteria whose members form a membrane-associated ribonucleoprotein (RNP) complex. This complex facilitates cellular adaptation to diverse stresses such as exposure to cold, short-chain alcohols, and elevated Mg2+ concentrations. Here, we report additional phenotypes exhibited by Halalkalibacterium halodurans (formerly called Bacillus halodurans) strains lacking functional OLE RNP complexes. Genetic disruption of the complex causes restricted growth compared to wild-type cells when cultured in minimal media (MM) wherein glucose is replaced with alternative carbon/energy sources. Genetic suppressor selections conducted in glutamate MM yielded isolates that carry mutations in or near genes relevant to Mn2+ homeostasis (ykoY and mntB), phosphate homeostasis (phoR), and putative multidrug resistance (bmrCD). These functional links between OLE RNA, carbon/energy management, and other fundamental processes including protein secretion are consistent with the hypothesis that the OLE RNP complex is a major contributor to cellular adaptation to unfavorable growth conditions.

A genome-wide CRISPR/Cas9 screen identifies a role for Rab5A and early endosomes in hepatitis E virus replication.

Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. As the other positive-strand RNA viruses, it is believed to replicate its genome in a membrane-associated replication complex. However, current understanding of the host factors required for productive HEV infection is limited and the site as well as the composition of the HEV replication complex are still poorly characterized. To identify host factors required for HEV RNA replication, we performed a genome-wide CRISPR/Cas9 screen in permissive human cell lines harboring subgenomic HEV replicons allowing for positive and negative selection. Among the validated candidates, Ras-related early endosomal protein Rab5A was selected for further characterization. siRNA-mediated silencing of Rab5A and its effectors APPL1 and EEA1, but not of the late and recycling endosome components Rab7A and Rab11A, respectively, significantly reduced HEV RNA replication. Furthermore, pharmacological inhibition of Rab5A and of dynamin-2, required for the formation of early endosomes, resulted in a dose-dependent decrease of HEV RNA replication. Colocalization studies revealed close proximity of Rab5A, the HEV ORF1 protein, corresponding to the viral replicase, as well as HEV positive- and negative-strand RNA. In conclusion, we successfully exploited CRISPR/Cas9 and selectable subgenomic replicons to identify host factors of a noncytolytic virus. This approach revealed a role for Rab5A and early endosomes in HEV RNA replication, likely by serving as a scaffold for the establishment of functional replication complexes. Our findings yield insights into the HEV life cycle and the virus-host interactions required for productive infection.

Peptide Epimerase Responsible for d-Amino Acid Introduction in Poly-γ-glutamic Acid Biosynthesis.

Poly-γ-glutamic acid (PGA) is a natural polymer of d- and/or l-glutamic acid (Glu) linked by isopeptide bonds. We recently showed that PGA synthetase, an enzyme complex composed of PgsB, PgsC, and PgsA, uses only l-Glu for polymerization, and d-Glu residues are introduced by peptide epimerization. However, it remains unclear which of the three enzymes is responsible for epimerization because in vitro functional characterization of the membrane-associated PgsBCA complex has never been successful. Here, we performed gene exchange experiments and showed that PgsA is responsible for the epimerization. Additionally, we identified a region in PgsA that modulates epimerization activity based on homology modeling from the recently solved structure of MslH, which showed 53% identity to PgsA. Our results suggested that d/l-ratios of the PGA product can be altered by introducing amino acid substitutions in this region, which will be useful for the production of PGA with controlled d/l-ratios.

Membrane extraction in native lipid nanodiscs reveals dynamic regulation of Cdc42 complexes during cell polarization

Embryonic development requires the establishment of cell polarity to enable cell fate segregation and tissue morphogenesis. This process is regulated by Par complex proteins, which partition into polarized membrane domains and direct downstream polarized cell behaviors. The kinase aPKC (along with its cofactor Par6) is a key member of this network and can be recruited to the plasma membrane by either the small GTPase Cdc42 or the scaffolding protein Par3. Although in vitro interactions among these proteins are well established, much is still unknown about the complexes they form during development. Here, to enable the study of membrane-associated complexes ex vivo, we used a maleic acid copolymer to rapidly isolate membrane proteins from single C. elegans zygotes into lipid nanodiscs. We show that native lipid nanodisc formation enables detection of endogenous complexes involving Cdc42, which are undetectable when cells are lysed in detergent. We found that Cdc42 interacts more strongly with aPKC/Par6 during polarity maintenance than polarity establishment, two developmental stages that are separated by only a few minutes. We further show that Cdc42 and Par3 do not bind aPKC/Par6 simultaneously, confirming recent in vitro findings in an ex vivo context. Our findings establish a new tool for studying membrane-associated signaling complexes and reveal an unexpected mode of polarity regulation via Cdc42.

Elucidation of the structural dynamics of mutations in PHB2 protein associated with growth suppression and cancer progression

Prohibitin is a multifunctional protein that plays an important role in numerous cellular processes. Membrane-associated mitochondrial prohibitin complex is made up of two subunits, PHB1 and PHB2 which are ubiquitously expressed and analogous to each other. High levels of prohibitin expression have consequently been found in esophageal cancer, endometrial adenocarcinoma, gastric cancer, hepatocellular carcinoma, breast cancer and bladder cancer. The aim of this study is to analyse two-point mutation PHB2_MT1(I → A) and PHB2_MT2(I → P), their effect on PHB2 protein and its effect on formation of mitochondrial complex. It is a residual level study, based on current experimental validation. To establish the effects of the two-point mutations, computational approaches such as molecular modelling, molecular docking, normal mode simulation, molecular dynamics simulations and MM/GBSA were used. An analysis of the energy dynamics of both unbound and complex proteins was conducted to elucidate how mutations impact the energy distribution of PHB2. Our study confirmed that the two mutations decreased the overall stability of PHB2. This was evidenced by heightened atomic fluctuations within the mutated region, accompanied by elevated deviations observed in RMSD and Rg values. Furthermore, these mutations were correlated with a decline in the organization of secondary structural elements. The mutations in PHB2_MT1 and PHB2_MT2 resulted in formation a less stable prohibitin complex. Thus, PHB1 and PHB2 may act as molecular target or novel biomarkers for therapeutic intervention in numerous forms of malignancies.

New structural insights provide a different angle on steroid sulfatase action

A central part of human sulfation pathways is the spatially and temporally controlled desulfation of biologically highly potent steroid hormones. The responsible enzyme - steroid sulfatase (STS) - is highly expressed in placenta and peripheral tissues, such as fat, colon, and the brain. The shape of this enzyme and its mechanism are probably unique in biochemistry. STS was believed to be a transmembrane protein, spanning the Golgi double-membrane by stem region formed by two extended internal alpha-helices. New crystallographic data however challenge this view. STS now is portraited as a trimeric membrane-associated complex. We discuss the impact of these results on STS function and sulfation pathways in general and we hypothesis that this new STS structural understanding suggests product inhibition to be a regulator of STS enzymatic activity.

The CTP-binding domain is disengaged from the DNA-binding domain in a cocrystal structure of Bacillus subtilis Noc–DNA complex

In Bacillus subtilis, a ParB-like nucleoid occlusion protein (Noc) binds specifically to Noc-binding sites (NBSs) on the chromosome to help coordinate chromosome segregation and cell division. Noc does so by binding to CTP to form large membrane-associated nucleoprotein complexes to physically inhibit the assembly of the cell division machinery. The site-specific binding of Noc to NBS DNA is a prerequisite for CTP-binding and the subsequent formation of a membrane-active DNA-entrapped protein complex. Here, we solve the structure of a C-terminally truncated B.subtilis Noc bound to NBS DNA to reveal the conformation of Noc at this crucial step. Our structure reveals the disengagement between the N-terminal CTP-binding domain and the NBS-binding domain of each DNA-bound Noc subunit; this is driven, in part, by the swapping of helices 4 and 5 at the interface of the two domains. Site-specific crosslinking data suggest that this conformation of Noc-NBS exists in solution. Overall, our results lend support to the recent proposal that parS/NBS binding catalyzes CTP binding and DNA entrapment by preventing the reengagement of the CTP-binding domain and the DNA-binding domain from the same ParB/Noc subunit.

Multifunctional Protein A Is the Only Viral Protein Required for Nodavirus RNA Replication Crown Formation.

Positive-strand RNA virus RNA genome replication occurs in membrane-associated RNA replication complexes (RCs). Nodavirus RCs are outer mitochondrial membrane invaginations whose necked openings to the cytosol are "crowned" by a 12-fold symmetrical proteinaceous ring that functions as the main engine of RNA replication. Similar protein crowns recently visualized at the openings of alphavirus and coronavirus RCs highlight their broad conservation and functional importance. Using cryo-EM tomography, we earlier showed that the major nodavirus crown constituent is viral protein A, whose polymerase, RNA capping, membrane interaction and multimerization domains drive RC formation and function. Other viral proteins are strong candidates for unassigned EM density in the crown. RNA-binding RNAi inhibitor protein B2 co-immunoprecipitates with protein A and could form crown subdomains that protect nascent viral RNA and dsRNA templates. Capsid protein may interact with the crown since nodavirus virion assembly has spatial and other links to RNA replication. Using cryoelectron tomography and complementary approaches, we show that, even when formed in mammalian cells, nodavirus RC crowns generated without B2 and capsid proteins are functional and structurally indistinguishable from mature crowns in infected Drosophila cells expressing all viral proteins. Thus, the only nodaviral factors essential to form functional RCs and crowns are RNA replication protein A and an RNA template. We also resolve apparent conflicts in prior results on B2 localization in infected cells, revealing at least two distinguishable pools of B2. The results have significant implications for crown structure, assembly, function and control as an antiviral target.

Molecular architecture of the Chikungunya virus replication complex.

To better understand how positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) to synthesize, process, and transport viral RNA in virus-infected cells, we determined both the high-resolution structure of the core RNA replicase of chikungunya virus and the native RC architecture in its cellular context at subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase, the viral polymerase nsP4, which is in complex with nsP2 helicase-protease, sits in the central pore of the membrane-anchored nsP1 RNA-capping ring. The addition of a large cytoplasmic ring next to the C terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses.

Characterization of Mayaro virus (strain BeAn343102) biology in vertebrate and invertebrate cellular backgrounds.

Mayaro virus (MAYV) is an emerging New World alphavirus (genus Alphavirus, family Togaviridae) that causes acute multiphasic febrile illness, skin rash, polyarthritis and occasional severe clinical phenotypes. The virus lifecycle alternates between invertebrate and vertebrate hosts. Here we characterize the replication features, cell entry, lifecycle and virus-related cell pathology of MAYV using vertebrate and invertebrate in vitro models. Electron-dense clathrin-coated pits in infected cells and reduced viral production in the presence of dynasore, ammonium chloride and bafilomycin indicate that viral entry occurs through pH-dependent endocytosis. Increase in FITC-dextran uptake (an indicator of macropinocytosis) in MAYV-infected cells, and dose-dependent infection inhibition by 5-(N-ethyl-N-isopropyl) amiloride (a macropinocytosis inhibitor), indicated that macropinocytosis is an additional entry mechanism of MAYV in vertebrate cells. Acutely infected vertebrate and invertebrate cells formed cytoplasmic or membrane-associated extracytoplasmic replication complexes. Mosquito cells showed modified hybrid cytoplasmic vesicles that supported virus replication, nucleocapsid production and maturation. Mature virus particles were released from cells by both exocytosis and budding from the cell membrane. MAYV replication was cytopathic and associated with induction of apoptosis by the intrinsic pathway, and later by the extrinsic pathway in infected vertebrate cells. Given that MAYV is expanding its geographical existence as a potential public health problem, this study lays the foundation for biological understanding that will be valuable for therapeutic and preventive interventions.

TRAF4 hyperactivates HER2 signaling and contributes to Trastuzumab resistance in HER2-positive breast cancer

The HER2 receptor modulates downstream signaling by forming homodimers and heterodimers with other members of the HER family. For patients with HER2-positive breast cancer, Trastuzumab, an anti-HER2 monoclonal antibody as first-line therapy has shown significant survival benefits. However, the development of acquired resistance to Trastuzumab continues to be a significant obstacle. TNF receptor-associated factor 4 (TRAF4) upregulation was discovered to be associated with a worse clinical outcome. Here we identified TRAF4 overexpression as one of the putative mechanisms for HER2-positive breast cancer cells to maintain HER2 signaling during Trastuzumab treatment, while TRAF4 knockdown reduced HER2 stability and improved Trastuzumab sensitivity. Mechanistically, TRAF4 regulates HER2 level through its impact on SMAD specific E3 ubiquitin protein ligase protein 2 (SMURF2). The development of a membrane-associated protein complex containing HER2, TRAF4, and SMURF2 has been observed. SMURF2 bound to the HER2 cytoplasmic domain, and directly ubiquitinated it leading to HER2 degradation, whereas TRAF4 stabilized HER2 by degrading SMURF2 and inhibiting the binding of SMURF2 to HER2. Moreover, downregulation of TRAF4 has decreased the AKT/mTOR signaling. In conclusion, we discovered a new HER2 signaling regulation that involves the TRAF4-SMURF2 complex, a possible mechanism that might contribute to anti-HER2 resistance, making TRAF4 a viable target for treating HER2 + breast cancer.

Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System.

ABSTRACTThe bacterial flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the flagellar motility machinery. Homologous nonflagellar (NF-T3SS) proteins form the injectisome machinery that bacteria use to deliver effector proteins into eukaryotic cells, and other family members were recently reported to be involved in the formation of membrane nanotubes. Here, we describe a novel, evolutionarily widespread, hat-shaped structure embedded in the inner membranes of bacteria, of yet-unidentified function, that is present in species containing fT3SS. Mutant analysis suggests a relationship between this novel structure and the fT3SS, but not the NF-T3SS. While the function of this novel structure remains unknown, we hypothesize that either some of the fT3SS proteins assemble within the hat-like structure, perhaps including the fT3SS core complex, or that fT3SS components regulate other proteins that form part of this novel structure.IMPORTANCE The type III secretion system (T3SS) is a fascinating suite of proteins involved in building diverse macromolecular systems, including the bacterial flagellar motility machine, the injectisome machinery that bacteria use to inject effector proteins into host cells, and probably membrane nanotubes which connect bacterial cells. Here, we accidentally discovered a novel inner membrane-associated complex related to the flagellar T3SS. Examining our lab database, which is comprised of more than 40,000 cryo-tomograms of dozens of species, we discovered that this novel structure is both ubiquitous and ancient, being present in highly divergent classes of bacteria. Discovering a novel, widespread structure related to what are among the best-studied molecular machines in bacteria will open new venues for research aiming at understanding the function and evolution of T3SS proteins.

Genome-wide association identifies a missing hydrolase for tocopherol synthesis in plants

The tocopherol biosynthetic pathway, encoded by VTE genes 1 through 6, is highly conserved in plants but most large effect quantitative trait loci for seed total tocopherols (totalT) lack VTE genes, indicating other activities are involved. A genome-wide association study of Arabidopsis seed tocopherols showed five of seven significant intervals lacked VTE genes, including the most significant, which mapped to an uncharacterized, seed-specific, envelope-localized, alpha/beta hydrolase with esterase activity, designated AtVTE7. Atvte7 null mutants decreased seed totalT 55% while a leaky allele of the maize ortholog, ZmVTE7, decreased kernel and leaf totalT 38% and 49%, respectively. Overexpressing AtVTE7 or ZmVTE7 partially or fully complemented the Atvte7 seed phenotype and increased leaf totalT by 3.6- and 6.9-fold, respectively. VTE7 has the characteristics of an esterase postulated to provide phytol from chlorophyll degradation for tocopherol synthesis, but bulk chlorophyll levels were unaffected in vte7 mutants and overexpressing lines. Instead, levels of specific chlorophyll biosynthetic intermediates containing partially reduced side chains were impacted and strongly correlated with totalT. These intermediates are generated by a membrane-associated biosynthetic complex containing protochlorophyllide reductase, chlorophyll synthase, geranylgeranyl reductase (GGR) and light harvesting-like 3 protein, all of which are required for both chlorophyll and tocopherol biosynthesis. We propose a model where VTE7 releases prenyl alcohols from chlorophyll biosynthetic intermediates, which are then converted to the corresponding diphosphates for tocopherol biosynthesis.

Expression, Localization, and Protein Interactions of the Partitioning Proteins in the Gonococcal Type IV Secretion System.

Partitioning proteins are well studied as molecular organizers of chromosome and plasmid segregation during division, however little is known about the roles partitioning proteins can play within type IV secretion systems. The single-stranded DNA (ssDNA)-secreting gonococcal T4SS has two partitioning proteins, ParA and ParB. These proteins work in collaboration with the relaxase TraI as essential facilitators of type IV secretion. Bacterial two-hybrid experiments identified interactions between each partitioning protein and the relaxase. Subcellular fractionation demonstrated that ParA is found in the cellular membrane, whereas ParB is primarily in the membrane, but some of the protein is in the soluble fraction. Since TraI is known to be membrane-associated, these data suggest that the gonococcal relaxosome is a membrane-associated complex. In addition, we found that translation of ParA and ParB is controlled by an RNA switch. Different mutations within the stem-loop sequence predicted to alter folding of this RNA structure greatly increased or decreased levels of the partitioning proteins.