Predicted Transmembrane Domains Research Articles

Identification and Characterization of the Stimulator of Interferon Genes (STING) in Chinese Giant Salamander Andrias davidianus

The stimulator of interferon genes (STING) is a central and multifaceted mediator in innate immunity, and plays vital roles in defending against pathogen infection. In this study, we identified and functionally characterized the STING homolog from the Chinese giant salamander Andrias davidianus (AdSTING) for the first time. The open reading frame of AdSTING encodes a 362 amino acid protein with a predicted molecular mass of 41.6 kDa, which shares 31.1–46.7% of its sequence identity with STING homologs in other vertebrates. Structural analysis revealed that AdSTING possesses four predicted transmembrane domains (TMs) at the N-terminal, and a C-terminal domain (CTD) featuring a dimerization domain (DD), a c-di-GMP-binding domain (CBD), and a short C-terminal tail (CTT). Tissue distribution analysis showed that AdSTING mRNA was ubiquitously expressed in all examined tissues, with abundant expression in muscles, intestine, and thymus. During Andrias davidianus ranavirus (ADRV) infection, significant up-regulation of AdSTING expression was observed in the thymus, spleen, and kidney. Upon different stimuli in vitro, the expression of AdSTING was significantly induced by ADRV infection or polyinosin-polycytidylic acid (poly I:C) stimulation, but no obvious changes were observed during lipopolysaccharide (LPS) stimulation. Subcellular localization analysis revealed that AdSTING mainly localized in the cytoplasm in the Chinese giant salamander thymus cell line (GSTC) and co-localized with the endoplasmic reticulum (ER). Luciferase reporter assays confirmed the ability of AdSTING to activate the interferon-stimulated response element (ISRE) and interferon (IFN) promoter. Furthermore, overexpression of AdSTING effectively decreased ADRV infection, as evidenced by the reduction of virus titers and viral gene expression. Collectively, our findings underscore the pivotal role of AdSTING in the antiviral innate immunity of the Chinese giant salamander, offering insights into the functional evolution of STING in amphibians.

A knockdown gene approach identifies an insect vector membrane protein with leucin-rich repeats as one of the receptors for the VmpA adhesin of flavescence dorée phytoplasma.

The adhesion of flavescence dorée phytoplasma to the midgut epithelium cells of their insect vectors is partially mediated by the variable membrane protein A (VmpA), an adhesin which shows lectin properties. In order to identify the insect receptor for VmpA, we identified Euscelidius variegatus cell proteins interacting with recombinant VmpA-His6. The E. variegatus proteins were identified by mass spectrometry analysis of VmpA-E. variegatus protein complexes formed upon in vitro interaction assays. To assess their impact in VmpA binding, we reduced the expression of the candidate genes on E. variegatus cells in culture by dsRNA-mediated RNAi. The effect of candidate gene knockdown on VmpA binding was measured by the capacity of E. variegatus cells to bind VmpA-coated fluorescent beads. There were 13 candidate proteins possessing potential N-glycosylation sites and predicted transmembrane domains selected. The decrease of expression of an unknown transmembrane protein with leucine-rich repeat domains (uk1_LRR) was correlated with the decreased adhesion of VmpA beads to E. variegatus cells. The uk1_LRR was more expressed in digestive tubes than salivary glands of E. variegatus. The protein uk1_LRR could be implicated in the binding with VmpA in the early stages of insect infection following phytoplasmas ingestion.

Regulation of acetate tolerance by small ORF-encoded polypeptides modulating efflux pump specificity in Methylomonas sp. DH-1

BackgroundMethanotrophs have emerged as promising hosts for the biological conversion of methane into value-added chemicals, including various organic acids. Understanding the mechanisms of acid tolerance is essential for improving organic acid production. WatR, a LysR-type transcriptional regulator, was initially identified as involved in lactate tolerance in a methanotrophic bacterium Methylomonas sp. DH-1. In this study, we investigated the role of WatR as a regulator of cellular defense against weak organic acids and identified novel target genes of WatR.ResultsBy conducting an investigation into the genome-wide binding targets of WatR and its role in transcriptional regulation, we identified genes encoding an RND-type efflux pump (WatABO pump) and previously unannotated small open reading frames (smORFs), watS1 to watS5, as WatR target genes activated in response to acetate. The watS1 to watS5 genes encode polypeptides of approximately 50 amino acids, and WatS1 to WatS4 are highly homologous with one predicted transmembrane domain. Deletion of the WatABO pump genes resulted in decreased tolerance against formate, acetate, lactate, and propionate, suggesting its role as an efflux pump for a wide range of weak organic acids. WatR repressed the basal expression of watS genes but activated watS and WatABO pump genes in response to acetate stress. Overexpression of watS1 increased tolerance to acetate but not to other acids, only in the presence of the WatABO pump. Therefore, WatS1 may increase WatABO pump specificity toward acetate, switching the general weak acid efflux pump to an acetate-specific efflux pump for efficient cellular defense against acetate stress.ConclusionsOur study has elucidated the role of WatR as a key transcription factor in the cellular defense against weak organic acids, particularly acetate, in Methylomonas sp. DH-1. We identified the genes encoding WatABO efflux pump and small polypeptides (WatS1 to WatS5), as the target genes regulated by WatR for this specific function. These findings offer valuable insights into the mechanisms underlying weak acid tolerance in methanotrophic bacteria, thereby contributing to the development of bioprocesses aimed at converting methane into value-added chemicals.

A single amino acid substitution in MdLAZY1A dominantly impairs shoot gravitropism in Malus.

Plant architecture is 1 of the most important factors that determines crop yield potential and productivity. In apple (Malus domestica), genetic improvement of tree architecture has been challenging due to a long juvenile phase and growth as complex trees composed of a distinct scion and a rootstock. To better understand the genetic control of apple tree architecture, the dominant weeping growth phenotype was investigated. We report the identification of MdLAZY1A (MD13G1122400) as the genetic determinant underpinning the Weeping (W) locus that largely controls weeping growth in Malus. MdLAZY1A is 1 of the 4 paralogs in apple that are most closely related to AtLAZY1 involved in gravitropism in Arabidopsis (Arabidopsis thaliana). The weeping allele (MdLAZY1A-W) contains a single nucleotide mutation c.584T>C that leads to a leucine to proline (L195P) substitution within a predicted transmembrane domain that colocalizes with Region III, 1 of the 5 conserved regions in LAZY1-like proteins. Subcellular localization revealed that MdLAZY1A localizes to the plasma membrane and nucleus in plant cells. Overexpressing the weeping allele in apple cultivar Royal Gala (RG) with standard growth habit impaired its gravitropic response and altered the growth to weeping-like. Suppressing the standard allele (MdLAZY1A-S) by RNA interference (RNAi) in RG similarly changed the branch growth direction to downward. Overall, the L195P mutation in MdLAZY1A is genetically causal for weeping growth, underscoring not only the crucial roles of residue L195 and Region III in MdLAZY1A-mediated gravitropic response but also a potential DNA base editing target for tree architecture improvement in Malus and other crops.

The cytoplasmic N-terminal tail of Zika virus NS4A protein forms oligomers in the absence of detergent or lipids

The non-structural (NS) NS4A protein in flaviviruses has three predicted transmembrane domains, is critical for virulence and participates in membrane morphogenesis. In Dengue virus (DENV), both hydrophylic N-terminal tail and its first transmembrane domain participate in the formation of oligomers which are important for pathogenicity. However, the relative importance of the N-terminal domain in oligomerization has been under debate. In particular, since in the absence of detergent or lipids, this domain (residues 1–48) in both DENV and Zika virus (ZIKV) NS4A, was found to be disordered. Recently, however, we reported preliminary data that showed that peptide ZIKV NS4A 4–58 adopts a defined secondary structure in aqueous solution and forms oligomers, signaling its importance for full length NS4A oligomerization. Herein we have performed detailed analytical ultracentrifugation experiments to further characterize the oligomerization of this peptide and also a shorter variant (residues 4–44). In both cases, sedimentation velocity produced a single species with concentration-dependent sedimentation coefficient, consistent with a fast equilibrium between at least two species. Combining sedimentation velocity and equilibrium experiments, data is best fitted to a monomer–dimer–trimer equilibrium. Possible models of NS4A oligomers obtained with AlphaFold-2 predict the stabilizing role for residues in this N-terminal domain, such as Arg20, Asn27, Ala44 and Glu50, all at highly conserved positions in flavivirus NS4A proteins. Our results are thus consistent with N-terminal domain interactions acting as one of the driving forces for NS4A homo-oligomerization.

Cysteine mutagenesis reveals novel structure-function features within the predicted transmembrane domain of chloride intracellular ion channel 1 (CLIC1).

Chloride intracellular ion channels (CLICs) are a unique class of metamorphic proteins that exists as soluble proteins and can auto-insert into the membrane to form an ion channel. The soluble structure of all the CLICs is well established but the structural information on the membrane form is lacking. CLICs are known to have a single putative transmembrane (PTM) region and proteins oligomerize to a tetramer to form a functional channel. CLIC1 is a sensor of oxidative stress, overexpressed in different types of cancer, and involved in diabetes, cell viability, and angiogenesis. The PTM region of CLIC1 encompasses residue 24-46, where cysteine at position 24 is a critical redox-sensitive residue. However, the precise arrangement of the residues in the pore region is not known. Hence, to identify the key residue involved in the gating mechanism and ion selectivity, the putative residues lining the pore region were mutated using site-directed mutagenesis. We incorporated the substituted cysteine accessibility method (SCAM) and recorded the channel activity of the mutants in the whole-cell and inside-out configurations. The ion selectivity was measured by perfusing with different anions (chloride, bromide, fluoride, and phosphate). The open channel probability (Po) is significantly higher in R29C mutant (0.88) compared to WT (0.21), suggesting R29C residue is involved in CLIC1 gating. Similarly, the CLIC1 is highly selective for chloride ions as compared to other anions, and the selective filer is located between V33C and K37C residues. We probed substituted cysteine residues with cadmium and identified that the T40C showed a reduction in Po and amplitude. Our results have delineated the pore lining of the CLIC1 and identified the residues involved in its gating and regulating the chloride selectivity.

Oropouche Virus Glycoprotein Topology and Cellular Requirements for Glycoprotein Secretion.

Oropouche virus (OROV; genus Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. We used recombinant expression of the OROV M polyprotein, which encodes the surface glycoproteins Gn and Gc plus the nonstructural protein NSm, to probe the cellular determinants for OROV assembly and budding. Gn and Gc self-assemble and are secreted independently of NSm. Mature OROV Gn has two predicted transmembrane domains that are crucial for glycoprotein translocation to the Golgi complex and glycoprotein secretion, and unlike related orthobunyaviruses, both transmembrane domains are retained during Gn maturation. Disruption of Golgi function using the drugs brefeldin A and monensin inhibits glycoprotein secretion. Infection studies have previously shown that the cellular endosomal sorting complexes required for transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. A dominant-negative form of the ESCRT-associated ATPase VPS4 significantly reduces recombinant OROV glycoprotein secretion and blocks virus release from infected cells, and VPS4 partly colocalizes with OROV glycoproteins and membranes costained with Golgi markers. Furthermore, immunoprecipitation and fluorescence microscopy experiments demonstrate that OROV glycoproteins interact with the ESCRT-III component CHMP6, with overexpression of a dominant-negative form of CHMP6 significantly reducing OROV glycoprotein secretion. Taken together, our data highlight differences in M polyprotein processing across orthobunyaviruses, indicate that Golgi and ESCRT function are required for glycoprotein secretion, and identify CHMP6 as an ESCRT-III component that interacts with OROV glycoproteins. IMPORTANCE Oropouche virus causes Oropouche fever, a debilitating illness common in South America that is characterized by high fever, headache, myalgia, and vomiting. The tripartite genome of this zoonotic virus is capable of reassortment, and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We developed a recombinant protein expression system to investigate the cellular determinants of OROV glycoprotein maturation and secretion. We show that the proteolytic processing of the M polypeptide, which encodes the surface glycoproteins (Gn and Gc) plus a nonstructural protein (NSm), differs between OROV and its close relative Bunyamwera virus. Furthermore, we demonstrate that OROV M glycoprotein secretion requires the cellular endosomal sorting complexes required for transport (ESCRT) membrane-remodeling machinery and identify that the OROV glycoproteins interact with the ESCRT protein CHMP6.

SARS-CoV-2 accessory protein 7b forms homotetramers in detergent

A global pandemic is underway caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome, like its predecessor SARS-CoV, contains open reading frames that encode accessory proteins involved in virus-host interactions active during infection and which likely contribute to pathogenesis. One of these accessory proteins is 7b, with only 44 (SARS-CoV) and 43 (SARS-CoV-2) residues. It has one predicted transmembrane domain fully conserved, which suggests a functional role, whereas most variability is contained in the predicted cytoplasmic C-terminus. In SARS-CoV, 7b protein is expressed in infected cells, and the transmembrane domain was necessary and sufficient for Golgi localization. Also, anti-p7b antibodies have been found in the sera of SARS-CoV convalescent patients. In the present study, we have investigated the hypothesis that SARS-2 7b protein forms oligomers with ion channel activity. We show that in both SARS viruses 7b is almost completely α-helical and has a single transmembrane domain. In SDS, 7b forms various oligomers, from monomers to tetramers, but only monomers when exposed to reductants. Combination of SDS gel electrophoresis and analytical ultracentrifugation (AUC) in both equilibrium and velocity modes suggests a dimer-tetramer equilibrium, but a monomer–dimer–tetramer equilibrium in the presence of reductant. This data suggests that although disulfide-linked dimers may be present, they are not essential to form tetramers. Inclusion of pentamers or higher oligomers in the SARS-2 7b model were detrimental to fit quality. Preliminary models of this association was generated with AlphaFold2, and two alternative models were exposed to a molecular dynamics simulation in presence of a model lipid membrane. However, neither of the two models provided any evident pathway for ions. To confirm this, SARS-2 p7b was studied using Planar Bilayer Electrophysiology. Addition of p7b to model membranes produced occasional membrane permeabilization, but this was not consistent with bona fide ion channels made of a tetrameric assembly of α-helices.

Molecular Characterization of Grass Carp GIPR and Effect of Nutrition States, Insulin, and Glucagon on Its Expression.

GIP plays an important regulatory role in glucose and lipid metabolism. As the specific receptor, GIPR is involved in this physiological process. To assess the roles of GIPR in teleost, the GIPR gene was cloned from grass carp. The ORF of cloned GIPR gene was 1560 bp, encoding 519 amino acids. The grass carp GIPR was the G-protein-coupled receptor which contains seven predicted transmembrane domains. In addition, two predicted glycosylation sites were contained in the grass carp GIPR. The grass carp GIPR expression is in multiple tissues and is highly expressed in the kidney, brain regions, and visceral fat tissue. In the OGTT experiment, the GIPR expression is markedly decreased in the kidney, visceral fat, and brain by treatment with glucose for 1 and 3 h. In the fast and refeeding experiment, the GIPR expression in the kidney and visceral fat tissue was significantly induced in the fast groups. In addition, the GIPR expression levels were markedly decreased in the refeeding groups. In the present study, the visceral fat accumulation of grass carp was induced by overfed. The GIPR expression was significantly decreased in the brain, kidney, and visceral fat tissue of overfed grass carp. In primary hepatocytes, the GIPR expression was promoted by treatment with oleic acid and insulin. The GIPR mRNA levels were significantly reduced by treatment with glucose and glucagon in the grass carp primary hepatocytes. To our knowledge, this is the first time the biological role of GIPR is unveiled in teleost.

Mitochondrial genomic analyses provide new insights into the “missing” atp8 and adaptive evolution of Mytilidae

BackgroundMytilidae, also known as marine mussels, are widely distributed in the oceans worldwide. Members of Mytilidae show a tremendous range of ecological adaptions, from the species distributed in freshwater to those that inhabit in deep-sea. Mitochondria play an important role in energy metabolism, which might contribute to the adaptation of Mytilidae to different environments. In addition, some bivalve species are thought to lack the mitochondrial protein-coding gene ATP synthase F0 subunit 8. Increasing studies indicated that the absence of atp8 may be caused by annotation difficulties for atp8 gene is characterized by highly divergent, variable length.ResultsIn this study, the complete mitochondrial genomes of three marine mussels (Xenostrobus securis, Bathymodiolus puteoserpentis, Gigantidas vrijenhoeki) were newly assembled, with the lengths of 14,972 bp, 20,482, and 17,786 bp, respectively. We annotated atp8 in the sequences that we assembled and the sequences lacking atp8. The newly annotated atp8 sequences all have one predicted transmembrane domain, a similar hydropathy profile, as well as the C-terminal region with positively charged amino acids. Furthermore, we reconstructed the phylogenetic trees and performed positive selection analysis. The results showed that the deep-sea bathymodiolines experienced more relaxed evolutionary constraints. And signatures of positive selection were detected in nad4 of Limnoperna fortunei, which may contribute to the survival and/or thriving of this species in freshwater.ConclusionsOur analysis supported that atp8 may not be missing in the Mytilidae. And our results provided evidence that the mitochondrial genes may contribute to the adaptation of Mytilidae to different environments.

Effects of individual amino acid mutations of zinc transporter ZIP8 on manganese- and cadmium-transporting activity

Zinc (Zn) transporter ZIP8, encoded by SLC39A8, is a unique transporter that can transport divalent manganese (Mn) and cadmium (Cd) in addition to Zn. Recently, associations between various human diseases and variant forms of ZIP8 have been reported. Four amino acid residues, V33, G38, S335, and I340, of human ZIP8 (hZIP8) are mutated in patients with congenital disorders of glycosylation (CDG), whose blood Mn levels are extremely low. Many genome-wide association studies have reported that the A391T mutation of hZIP8 caused by rs13107325 is associated with a wide range of diseases. However, the roles of individual mutations of hZIP8 on metal-transporting activity remain elusive. We established DT40 cells respectively expressing the four mutant hZIP8s and compared the Mn- and Cd-transporting activity between the mutants and wild-type hZIP8. Among the four mutations observed in the ZIP8-mutated CDG patients, the S335T and I340 N mutations in the predicted transmembrane domain 5 (TMD5) completely abolished Mn- and Cd-transporting activity, while V33 M or G35R mutations at the N-terminus did not. We also examined the A391T mutation, which slightly reduced metal transporting activity. Finally, we examined the effects of artificial mutations in the metal-binding motif EEXXH in the TMD5. Replacing EEXXH with HEXXH, which exists in most ZIP transporters, abolished the Mn- and Cd-transporting activity of hZIP8, indicating that glutamic acid in this motif plays a critical role in the unique affinity of ZIP8 for Mn and Cd. Thus, the utilization of DT40 cells enabled us to clarify the different functions of each residue of hZIP8 on metal transport.

Evolutionary diversification of the autophagy-related ubiquitin-like conjugation systems

ABSTRACT Two autophagy-related (ATG) ubiquitin-like conjugation systems, the ATG12 and ATG8 systems, play important roles in macroautophagy. While multiple duplications and losses of the ATG conjugation system proteins are found in different lineages, the extent to which the underlying systems diversified across eukaryotes is not fully understood. Here, in order to understand the evolution of the ATG conjugation systems, we constructed a transcriptome database consisting of 94 eukaryotic species covering major eukaryotic clades and systematically identified ATG conjugation system components. Both ATG10 and the C-terminal glycine of ATG12 are essential for the canonical ubiquitin-like conjugation of ATG12 and ATG5. However, loss of ATG10 or the C-terminal glycine of ATG12 occurred at least 16 times in a wide range of lineages, suggesting that possible covalent-to-non-covalent transition is not limited to the species that we previously reported such as Alveolata and some yeast species. Some species have only the ATG8 system (with conjugation enzymes) or only ATG8 (without conjugation enzymes). More than 10 species have ATG8 homologs without the conserved C-terminal glycine, and Tetrahymena has an ATG8 homolog with a predicted transmembrane domain, which may be able to anchor to the membrane independent of the ATG conjugation systems. We discuss the possibility that the ancestor of the ATG12 and ATG8 systems is more similar to ATG8. Overall, our study offers a whole picture of the evolution and diversity of the ATG conjugation systems among eukaryotes, and provides evidence that functional diversifications of the systems are more common than previously thought. Abbreviations: APEAR: ATG8–PE association region; ATG: autophagy-related; LIR: LC3-interacting region; NEDD8: neural precursor cell expressed, developmentally down-regulated gene 8; PE: phosphatidylethanolamine; SAMP: small archaeal modifier protein; SAR: Stramenopiles, Alveolata, and Rhizaria; SMC: structural maintenance of chromosomes; SUMO: small ubiquitin like modifier; TACK: Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota; UBA: ubiquitin like modifier activating enzyme; UFM: ubiquitin fold modifier; URM: ubiquitin related modifier.

Identification of CCp5 and FNPA as Novel Non-canonical Members of the CCp Protein Family in Babesia bovis.

Bovine babesiosis, caused by Babesia bovis, is an economically significant tick-borne disease that imposes restrictions to livestock production worldwide. Current methods to control bovine babesiosis have severe limitations and novel approaches, including transmission-blocking vaccines, are needed. Members of the widely conserved CCp family are multidomain adhesion proteins containing LCCL motifs, which are differentially expressed on gametocytes of apicomplexans, including Babesia spp. and Plasmodium spp. While Plasmodium parasites contain 6 distinct CCp genes, only three members (CCp 1-3) were previously identified in B. bovis. In this study, we describe the identification and characterization of two novel non-canonical members of the CCp gene family in B. bovis, named CCp5 and FNPA. The genes were identified in silico by TBLASTN using P. falciparum CCp family domains as queries. Unlike CCp1-3, the B. bovis CCp5 and FNPA proteins lack the LCCL canonical domain but contain other typical multidomain adhesion motifs which are present in classical CCp proteins. In addition, the B. bovis CCp5 and FNPA are in synteny with known CCp genes in related apicomplexans. Sequence analysis of these two proteins demonstrated high sequence conservation among B. bovis different isolates. Transcription, immunoblot, and immunofluorescence analyses demonstrated expression of CCp5 and FNPA in blood and in vitro induced sexual stages of B. bovis. The FNPA, in contrast to CCp5, has a predicted transmembrane domain, suggesting that it might be expressed in the surface of sexual stage parasites. Altogether, finding of this study support FNPA as a possible target of a transmission-blocking vaccine against B. bovis.

An extracellular matrix damage sensor signals through membrane-associated kinase DRL-1 to mediate cytoprotective responses in Caenorhabditis elegans.

We and others previously identified circumferential bands of collagen named annular furrows as key components of a damage sensor in the cuticle of Caenorhabditis elegans that regulates cytoprotective genes. Mutation or loss of noncollagen secreted proteins OSM-7, OSM-8, and OSM-11 activate the same cytoprotective responses without obvious changes to the cuticle indicating that other extracellular proteins are involved. Here, we used RNAi screening to identify protein kinase DRL-1 as a key modulator of cytoprotective gene expression and stress resistance in furrow and extracellular OSM protein mutants. DRL-1 functions downstream from furrow disruption and is expressed in cells that induce cytoprotective genes. DRL-1 is not required for the expression of cytoprotective genes under basal or oxidative stress conditions consistent with specificity to extracellular signals. DRL-1 was previously shown to regulate longevity via a "Dietary Restriction-Like" state, but it functions downstream from furrow disruption by a distinct mechanism. The kinase domain of DRL-1 is related to mammalian MEKK3, and MEKK3 is recruited to a plasma membrane osmosensor complex by a scaffold protein. In C. elegans, DRL-1 contains an atypical hydrophobic C-terminus with predicted transmembrane domains and is constitutively expressed at or near the plasma membrane where it could function to receive extracellular damage signals for cells that mount cytoprotective responses.

Multiple mutations in the predicted transmembrane domains of the cellulose synthase 3 (CesA3) of Phytophthora capsici can confer semi-dominant resistance to carboxylic acid amide fungicides

The current study established a clearer understanding of the molecular basis for resistance to carboxylic acid amide (CAA) fungicides. Although four cellulose synthase (CesA) genes were investigated, only F1073L, G1105A, V1109L in CesA3 were found to link to CAA-resistance in Phytophthora capsici. Back-transformation experiments confirmed the role of the three mutations in CAA-resistance. Inheritance studies also confirmed the link and indicated the resistance was semi-dominant with the heterozygous F1 and F2 progeny exhibiting intermediate resistance levels compared to the homozygous parents, which was validated by the pyrosequencing results. The semi-dominant nature of CAA-resistance implies that it could be easy for resistance to spread once resistance emerged, being facilitated by both sexual and asexual reproduction. Bioinformatic analysis indicated all mutations occurred in either the first or second of the predicted transmembrane domains at C-terminus of CesA3. Resistant isolates bearing different combinations of mutations were found to exhibit different resistance levels to different CAAs, indicating that each mutation could make different contributions to resistance phenotype depending on structural differences in different CAAs. The current results highlight the complex combinations of mutations and resistance phenotype, and further reinforces the research necessity to completely characterize CAA-resistance to develop appropriate strategies to manage resistance development.

Arabidopsis ADR1 helper NLR immune receptors localize and function at the plasma membrane in a phospholipid dependent manner.

Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive. We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM. Our results show that PM localization and stability of some RNLs and one CC-type NLR(CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function. We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM.

Unusual features and localization of the membrane kinome of Trypanosoma brucei.

In many eukaryotes, multiple protein kinases are situated in the plasma membrane where they respond to extracellular ligands. Ligand binding elicits a signal that is transmitted across the membrane, leading to activation of the cytosolic kinase domain. Humans have over 100 receptor protein kinases. In contrast, our search of the Trypanosoma brucei kinome showed that there were only ten protein kinases with predicted transmembrane domains, and unlike other eukaryotic transmembrane kinases, seven are predicted to bear multiple transmembrane domains. Most of the ten kinases, including their transmembrane domains, are conserved in both Trypanosoma cruzi and Leishmania species. Several possess accessory domains, such as Kelch, nucleotide cyclase, and forkhead-associated domains. Surprisingly, two contain multiple regions with predicted structural similarity to domains in bacterial signaling proteins. A few of the protein kinases have previously been localized to subcellular structures such as endosomes or lipid bodies. We examined the localization of epitope-tagged versions of seven of the predicted transmembrane kinases in T. brucei bloodstream forms and show that five localized to the endoplasmic reticulum. The last two kinases are enzymatically active, integral membrane proteins associated with the flagellum, flagellar pocket, or adjacent structures as shown by both fluorescence and immunoelectron microscopy. Thus, these kinases are positioned in structures suggesting participation in signal transduction from the external environment.

Identification of Key Amino Acids that Impact Organic Solute Transporter α/β (OSTα/β).

Organic solute transporter α/β (OSTα/β) is a bidirectional bile acid transporter localized on the basolateral membrane of hepatic, intestinal, and renal epithelial cells. OSTα/β plays a critical role in intestinal bile acid reabsorption and is upregulated in hepatic diseases characterized by elevated bile acids, whereas genetic variants in SLC51A/B have been associated with clinical cholestasis. OSTα/β also transports and is inhibited by commonly used medications. However, there is currently no high-resolution structure of OSTα/β, and structure-function data for OSTα, the proposed substrate-binding subunit, are lacking. The present study addressed this knowledge gap and identified amino acids in OSTα that are important for bile acid transport. This was accomplished using computational modeling and site-directed mutagenesis of the OSTα subunit to generate OSTα/β mutant cell lines. Out of the 10 OSTα/β mutants investigated, four (S228K, T229S, Q269E, Q269K) exhibited decreased [3H]-taurocholate (TCA) uptake (ratio of geometric means relative to OSTα/β wild type (WT) of 0.76, 0.75, 0.79, and 0.13, respectively). Three OSTα/β mutants (S228K, Q269K, E305A) had reduced [3H]-TCA efflux % (ratio of geometric means relative to OSTα/β WT of 0.86, 0.65, and 0.79, respectively). Additionally, several OSTα/β mutants demonstrated altered expression and cellular localization when compared with OSTα/β WT. In summary, we identified OSTα residues (Ser228, Thr229, Gln269, Glu305) in predicted transmembrane domains that affect expression of OSTα/β and may influence OSTα/β-mediated bile acid transport. These data advance our understanding of OSTα/β structure/function and can inform future studies designed to gain further insight into OSTα/β structure or to identify additional OSTα/β substrates and inhibitors. SIGNIFICANCE STATEMENT: OSTα/β is a clinically important transporter involved in enterohepatic bile acid recycling with currently no high-resolution protein structure and limited structure-function data. This study identified four OSTα amino acids (Ser228, Thr229, Gln269, Glu305) that affect expression of OSTα/β and may influence OSTα/β-mediated bile acid transport. These data can be utilized to inform future investigation of OSTα/β structure and refine molecular modeling approaches to facilitate the identification of substrates and/or inhibitors of OSTα/β.

Isolation and Characterization of Endomycorrhizal Fungi Associated with Growth Promotion of Blueberry Plants.

Despite their notable root mutualism with blueberries (Vaccinium spp.), studies related to Ericoid mycorrhizal (ERM) are relatively limited. In this study, we report the isolation of 14 endomycorrhizal fungi and their identification by fungal colony morphology characterization combined with PCR-amplified fungal internal transcribed spacer (ITS) sequence analyses. Six of the isolated strains were confirmed as beneficial mycorrhizal fungi for blueberry plants following inoculation. We observed the formation of typical ERM hyphae coil structures—which promote and nutritionally support growth—in blueberry seedlings and significant nitrogen and phosphorous content increases in diverse tissues. QRT-PCRs confirmed changes in VcPHT1s expression patterns. After the formation of ERM, PHT1-1 transcription in roots was upregulated by 1.4- to threefold, whilst expression of PHT1-3 and PHT1-4 in roots were downregulated 72% and 60%, respectively. Amino acid sequence analysis of all four VcPHT1s genes from the blueberry variety “Sharpblue” revealed an overall structural similarity of 67% and predicted transmembrane domains. Cloning and overexpression of PHT1-1 and PHT1-3 genes in transgenic Arabidopsis thaliana plants significantly enriched total phosphorus and chlorophyll content, confirming that PHT1-1 and PHT1-3 gene functions are associated with the transport and absorption of phosphorus.

Characterization of the gene family encoding metal tolerance proteins in Triticum urartu: Phylogenetic, transcriptional, and functional analyses.

Homeostasis of microelements in organisms is vital for normal metabolism. In plants, the cation diffusion facilitator (CDF) protein family, also known as metal tolerance proteins (MTPs), play critical roles in maintaining trace metal homeostasis. However, little is known about these proteins in wheat. In this study, we characterized the MTP family of Triticum urartu, the donor of 'A' genome of Triticum aestivum, and analysed their phylogenetic relationships, sequence signatures, spatial expression patterns in the diploid wheat, and their transport activity when heterologously expressed. Nine MTPs were identified in the T. urartu genome database, and were classified and designated based on their sequence similarity to Arabidopsis thaliana (Arabidopsis) and Oryza sativa MTPs. Phylogenetic and sequence analyses indicated that the triticum urartu metal tolerance protein (TuMTP)s comprise three Zn-CDFs, two Fe/Zn-CDFs, and four Mn-CDFs; and can be further classified into six subgroups. Among the TuMTPs, there are no MTP2-5 and MTP9-10 counterparts but two MTP1/8/11 orthologs in relation to AtMTPs. It was also shown that members of the same cluster share similar sequence characteristic, i.e. number of introns, predicted transmembrane domains, and motifs. When expressed in yeast, TuMTP1 and TuMTP1.1 conferred tolerance to Zn and Co but not to other metal ions; while TuMTP8, TuMTP8.1, TuMTP11, and TuMTP11.1 conferred tolerance to Mn. When expressed in Arabidopsis, TuMTP1 localized to the tonoplast and significantly enhanced Zn and Co tolerance. TuMTPs showed diverse tissue-specific expression patterns. Taken together, the closely clustered TuMTPs share structural features and metal specificity but play diverse roles in the homeostasis of microelements in plant cells.