C-terminal Green Fluorescent Protein Research Articles

Oryza-Specific Orphan Protein Triggers Enhanced Resistance to Xanthomonas oryzae pv. oryzae in Rice.

All genomes carry lineage-specific orphan genes lacking homology in their closely related species. Identification and functional study of the orphan genes is fundamentally important for understanding lineage-specific adaptations including acquirement of resistance to pathogens. However, most orphan genes are of unknown function due to the difficulties in studying them using helpful comparative genomics. Here, we present a defense-related Oryza-specific orphan gene, Xio1, specifically induced by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) in an immune receptor XA21-dependent manner. Salicylic acid (SA) and ethephon (ET) also induced its expression, but methyl jasmonic acid (MeJA) reduced its basal expression. C-terminal green fluorescent protein (GFP) tagged Xio1 (Xio1-GFP) was visualized in the nucleus and the cytosol after polyethylene glycol (PEG)-mediated transformation in rice protoplasts and Agrobacterium-mediated infiltration in tobacco leaves. Transgenic rice plants overexpressing Xio1-GFP showed significantly enhanced resistance to Xoo with reduced lesion lengths and bacterial growth, in company with constitutive expression of defense-related genes. However, all of the transgenic plants displayed severe growth retardation and premature death. Reactive oxygen species (ROS) was significantly produced in rice protoplasts constitutively expressing Xio1-GFP. Overexpression of Xio1-GFP in non-Oryza plant species, Arabidopsis thaliana, failed to induce growth retardation and enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. Our results suggest that the defense-related orphan gene Xio1 plays an important role in distinctive mechanisms evolved within the Oryza and provides a new source of Oryza-specific genes for crop-breeding programs.

Expression of actively soluble antigen-binding fragment (Fab) antibody and GFP fused Fab in the cytoplasm of the engineered Escherichia coli.

The expression of recombinant antibody fragments in the cytoplasmic space of Escherichia coli and the refolding process for restoring the structure and activity of such antibodies are not efficient. Herein, fragment antigen-binding (Fab) antibodies against miroestrol and deoxymiroestrol (MD-Fab) and their fusions with a green fluorescent protein (GFP) were expressed. The reactive MD-Fabs were successfully expressed as soluble and active forms in the cytoplasm of the SHuffle® T7 E. coli strain. Regarding the construct of MD-Fab alone, VH-CH1 could associate VL-CL into Fab in the oxidizing cytoplasm of the E. coli strain, and no additional in vitro refolding was needed. In the case of the fusions with GFP, when the C-terminus of VH-CH1 was linked with the N-terminus of GFP, the MD-Fab binding reactivity was retained, but the fluorescent activity of GFP interfered. When the C-terminus of GFP was linked to the N-terminus of VL-CL, the binding activity of MD-Fab was not observed. The constructed MD-Fabs had higher specificity toward deoxymiroestrol than the parental monoclonal antibody clone 12G11. In conclusion, MD-Fabs could be expressed using SHuffle® T7 E. coli cells. This process could be considered an economical, productive, and effective method to produce antibody fragments for immunoassay techniques.

Dissection of Cell Death Induction by Wheat Stem Rust Resistance Protein Sr35 and Its Matching Effector AvrSr35.

Nucleotide-binding leucine-rich repeat receptors (NLRs) are the most abundant type of immune receptors in plants and can trigger a rapid cell-death (hypersensitive) response upon sensing pathogens. We previously cloned the wheat NLR Sr35, which encodes a coiled-coil (CC) NLR that confers resistance to the virulent wheat stem rust race Ug99. Here, we investigated Sr35 signaling after Agrobacterium-mediated transient expression in Nicotiana benthamiana. Expression of Sr35 in N. benthamiana leaves triggered a mild cell-death response, which is enhanced in the autoactive mutant Sr35 D503V. The N-terminal tagging of Sr35 with green fluorescent protein (GFP) blocked the induction of cell death, whereas a C-terminal GFP tag did not. No domain truncations of Sr35 generated cell-death responses as strong as the wild type, but a truncation including the NB-ARC (nucleotide binding adaptor) shared by APAF-1, R proteins, and CED-4 domains in combination with the D503V autoactive mutation triggered cell death. In addition, coexpression of Sr35 with the matching pathogen effector protein AvrSr35 resulted in robust cell death and electrolyte leakage levels that were similar to autoactive Sr35 and significantly higher than Sr35 alone. Coexpression of Sr35-CC-NB-ARC and AvrSr35 did not induce cell death, confirming the importance of the leucine-rich repeat (LRR) domain for AvrSr35 recognition. These findings were confirmed through Agrobacterium-mediated transient expression in barley. Taken together, these results implicate the CC-NB-ARC domains of Sr35 in inducing cell death and the LRR domain in AvrSr35 recognition.

Grapevine fatty acid hydroperoxide lyase generates actin-disrupting volatiles and promotes defence-related cell death.

Fatty acid hydroperoxides can generate short-chained volatile aldehydes that may participate in plant defence. A grapevine hydroperoxide lyase (VvHPL1) clustering to the CYP74B class was functionally characterized with respect to a role in defence. In grapevine leaves, transcripts of this gene accumulated rapidly to high abundance in response to wounding. Cellular functions of VvHPL1 were investigated upon heterologous expression in tobacco BY-2 cells. A C-terminal green fluorescent protein (GFP) fusion of VvHPL1 was located in plastids. The overexpression lines were found to respond to salinity stress or the bacterial elicitor harpin by increasing cell death. This signal-dependent mortality response was mitigated either by addition of exogenous jasmonic acid or by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. By feeding different substrates to recombinantly expressed enzyme, VvHPL1 could also be functionally classified as true 13-HPL. The cognate products generated by this 13-HPL were cis-3-hexenal and trans-2-hexenal. Using a GFP-tagged actin marker line, one of these isomeric products, cis-3-hexenal, was found specifically to elicit a rapid disintegration of actin filaments. This response was not only observed in the heterologous system (tobacco BY-2), but also in a grapevine cell strain expressing this marker, as well as in leaf discs from an actin marker grape used as a homologous system. These results are discussed in the context of a role for VvHPL1 in a lipoxygenase-dependent signalling pathway triggering cell death-related defence that bifurcates from jasmonate-dependent basal immunity.

Functional severity of CLCNKB mutations correlates with phenotypes in patients with classic Bartter's syndrome.

The highly variable phenotypes observed in patients with classic Bartter's syndrome (BS) remain unsatisfactorily explained. The wide spectrum of functional severity of CLCNKB mutations may contribute to the phenotypic variability, and the genotype-phenotype association has not been established. Low-level expression of the human ClC-Kb channel in mammalian cells impedes the functional study of CLCNKB mutations, and the underlying cause is still unclear. The human ClC-Kb channel is highly degraded by proteasome in human embryonic kidney cells. The C-terminal in-frame green fluorescent protein fusion may slow down the proteasome-mediated proteolysis. Barttin co-expression necessarily improves the stability, membrane trafficking and gating of ClC-Kb. CLCNKB mutations in barttin-binding sites, dimer interface or selectivity filter often have severe functional consequences. The remaining chloride conductance of the ClC-Kb mutant channel significantly correlates with the phenotypes, such as age at diagnosis, plasma chloride concentration, and the degree of calciuria in patients with classic BS. Mutations in the CLCNKB gene encoding the human voltage-gated chloride ClC-Kb (hClC-Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests with highly variable phenotypes. The functional severity of the mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC-Kb in heterologous expression systems, the functional consequences of mutant channels have not been thoroughly examined, and the genotype-phenotype association has not been established. In this study, we found that hClC-Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In-frame fusion of green fluorescent protein (GFP) to the C-terminus of the channel may ameliorate proteasome degradation. Co-expression of barttin increased protein abundance and membrane trafficking of hClC-Kb and markedly increased functional chloride current. We then functionally characterized 18 missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC-Kb-GFP. Most CLCNKB mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin binding sites, dimer interface and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well-described functional consequences and clinical presentations for genotype-phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate. In conclusion, hClC-Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C-terminus of hClC-Kb improves protein expression. The functional severity of the CLCNKB mutation is an important determinant of the phenotype in classic BS.

Novel silica-forming peptides derived from Ectocarpus siliculosus

Abstract A synthetic R5 peptide showed the silica precipitation activity when added to silicic acid solution under ambient conditions. However, R5 peptide showed no silica precipitation activity below pH 7, an important pH range for biological applications. Here, we reported new silica-forming peptides (SFP), named EctP1 and EctP2. They showed better silica deposition ability than the R5 peptide. Particularly, at pH 6, EctP1 showed silica deposition activity while R5 did not. EctP1, EctP2, and R5 were genetically fused to the C-terminus of green fluorescent protein (GFP). GFP-SFP fusion proteins showed silicification ability, and GFP-EctP1 fusion protein showed the capacious silicification activity at wide pH range. The GFP-EctP2 fusion proteins showed higher protein expression levels than other fusion proteins. Furthermore, silicified GFP-SFP fusion protein exhibited an organic-inorganic complex form. These results indicate that the SFP fusion system is a novel tool for immobilizing biomolecules on silica material for biological and industrial applications.

A short motif in Arabidopsis CDK inhibitor ICK1 decreases the protein level, probably through a ubiquitin-independent mechanism.

The ICK/KRP family of cyclin-dependent kinase (CDK) inhibitors modulates the activity of plant CDKs through protein binding. Previous work has shown that changing the levels of ICK/KRP proteins by overexpression or downregulation affects cell proliferation and plant growth, and also that the ubiquitin proteasome system is involved in degradation of ICK/KRPs. We show in this study that the region encompassing amino acids 21 to 40 is critical for ICK1 levels in both Arabidopsis and yeast. To determine how degradation of ICK1 is controlled, we analyzed the accumulation of hemagglutinin (HA) epitope-tagged ICK1 proteins in yeast mutants defective for two ubiquitin E3 ligases. The highest level of HA-ICK1 protein was observed when both the N-terminal 1-40 sequence was removed and the SCF (SKP1-Cullin1-F-box complex) function disrupted, suggesting the involvement of both SCF-dependent and SCF-independent mechanisms in the degradation of ICK1 in yeast. A short motif consisting of residues 21-30 is sufficient to render green fluorescent protein (GFP) unstable in plants and had a similar effect in plants regardless of whether it was fused to the N-terminus or C-terminus of GFP. Furthermore, results from a yeast ubiquitin receptor mutant rpn10Δ indicate that protein ubiquitination is not critical in the degradation of GFP-ICK1(1-40) in yeast. These results thus identify a protein-destabilizing sequence motif that does not contain a typical ubiquitination residue, suggesting that it probably functions through an SCF-independent mechanism.

Identification and characterization of Candida utilis multidrug efflux transporter CuCdr1p.

The edible, nitrate assimilating, yeast Candida utilis is a commercial food additive, and it is a potentially useful host for heterologous protein expression. A number of ATP-binding cassette (ABC) transporters are multidrug efflux pumps that can cause multidrug resistance in opportunistic pathogens. In order to develop optimal novel antimicrobial agents it is imperative to understand the structure, function and expression of these transporters. With the ultimate aim of developing an alternative yeast host for the heterologous expression of eukaryotic membrane transporters, and to identify ABC transporters potentially associated with C. utilis multidrug resistance, we classified the entire repertoire of 30 C. utilis ABC proteins. We named the open reading frame most similar to the archetype multidrug efflux pump gene C. albicans CDR1 as CuCDR1 Overexpression of CuCDR1 in Saccharomyces cerevisiae ADΔ caused multidrug resistance similar to that of cells overexpressing CaCDR1 Unlike CaCdr1p, however, the C-terminally green fluorescent protein (GFP) tagged CuCdr1p-GFP was functionally impaired and did not properly localize to the plasma membrane. CuCdr1p function could be recovered however by adding a 15 amino acid linker -GAGGSAGGSGGAGAG- between CuCdr1p and the C-terminal GFP tag.

ATP-binding cassette transporter enhances tolerance to DDT in Tetrahymena.

The reuse of dichlorodiphenyltrichloroethane (DDT) as an indoor residual spray was permitted by the World Health Organization in 2007, and approximately 14 countries still use DDT to control disease vectors. The extensive exposure of insects to DDT has resulted in the emergence of DDT resistance, especially in mosquitoes, and the mechanism for this resistance in mosquitoes has been widely reported. Spraying can also introduce DDT directly into surface water, and DDT can subsequently accumulate in microorganisms, but the mechanism for the resistance to DDT degradation in microorganisms is unclear. Using whole-genome microarray analysis, we detected an abcb15 gene that was up-regulated in a specific manner by DDT treatment in T. thermophile. The deduced ABCB15 peptide sequence had two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs) to form the structure TMD-NBD-TMD-NBD, and each NBD contained three conserved motifs: Walker-A, C-loop, and Walker-B, which indicated the T. thermophila abcb15 was a typical ABC transporter gene. The expression of ABCB15 fused with a C-terminal green fluorescent protein was found to be on the periphery of the cell, suggesting that ABCB15 was a membrane pump protein. In addition, cells with abcb15 partially knocked down (abcb15-KD) grew slower than wild-type cells in the presence of 256 mg L(-1) DDT, indicating the tolerance of abcb15-KD strain to DDT exposure was decreased. Thus, we suggest that in Tetrahymena, the membrane pump protein encoded by ABCT gene abcb15 can enhance the tolerance to DDT and protect cells from this exogenous toxin by efficiently pumping it to the extracellular space.

Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex.

Green fluorescent protein (GFP), fused to the N or C terminus of a protein of interest, is widely used to monitor the localization and mobility of proteins in cells. RAD51 is an essential protein that functions in mitotic DNA repair and meiotic chromosome segregation by promoting the homologous recombination reaction. A previous genetic study with Arabidopsis thaliana revealed that GFP fused to the C terminus of RAD51 (RAD51-GFP) inhibits mitotic DNA repair, but meiotic homologous recombination remained unaffected. To determine how the C-terminal GFP specifically inhibits mitotic DNA repair by RAD51, we purified rice RAD51A1-GFP and RAD51A2-GFP, and performed biochemical analyses. Interestingly, purified RAD51A1-GFP and RAD51A2-GFP are proficient in DNA binding and ATP hydrolysis. However, nucleoprotein complexes containing single-stranded DNA and RAD51A1-GFP or RAD51A2-GFP are significantly defective in binding to the second DNA molecule (secondary DNA binding), and consequently fail to catalyze homologous pairing. In contrast, RAD51A1-GFP and RAD51A2-GFP efficiently stimulated homologous pairing promoted by the meiosis-specific RAD51 isoform DMC1. These biochemical characteristics are well conserved in human RAD51-GFP. Therefore, GFP fused to the C terminus of RAD51 abolishes the homologous pairing activity of RAD51 by disrupting secondary DNA binding, but does not affect its DMC1-stimulating activity.

A Family of HCN Channel Homologs in Bacteria

HCN channels belong to the superfamily of six transmembrane domain voltage-gated ion channels. Functionally, these channels activate upon membrane hyperpolarization and carry an inward current that is weakly selective between potassium and sodium and can be modulated by cyclic nucleotides. At the structural level, insight into these channels is limited to a crystal structure of the intracellular C-linker and cyclic nucleotide binding domain, which assembles into a tetramer. To advance our structural understanding of HCN channels, we investigated bacterial homologs of HCN channels as candidates for large-scale expression and future structural studies. Using a BLAST search of the bacterial genome database we identified different homologs, which have a sequence identity of 24-32% with the human HCN2 channel. Using a C-terminal fusion with green fluorescent protein (GFP) we investigated whether the bacterial HCN homologs could be expressed in E. coli. We identified homologs that could be expressed at a whole-cell fluorescence level of ∼40% compared to KcsA-GFP as a positive control. Next, we conducted detergent screening using fluorescence size exclusion chromatography (FSEC). We found that dodecylmaltoside or undecylmaltoside are suitable detergents to solubilize bacterial HCN channels in a tetrameric monodisperse state.In parallel, we investigated the functional properties of the bacterial HCN channel homologs using expression of C-terminal GFP fusions in Xenopus oocytes. Using cell-attached patch recordings we observed in a minority of patches a current that may closely resemble the kinetics of the invertebrate spHCN channel as clear current inactivation is observed at most hyperpolarizing potentials. Confocal microscopy demonstrates marked fluorescence just below the oocyte membrane, indicating that trafficking to the oocyte cell membrane may be compromised and limits our success in obtaining patches for detailed functional characterization.Together, our biochemical characterization paves the way for large-scale production and crystallization screening.

Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase.

Protein disulfide isomerases (PDIs) catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER). Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′) and two non-catalytic domains (b, b′), in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6). Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11) possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP) fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6) functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis

Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-l-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for >85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H2O2), but only ~33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H2O2 under untreated conditions.

Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC, a Receptor Protein of Quorum Sensing in Staphylococcus aureus

Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP) fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) at yields of ≥10 mg/L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-(23)-lauryl-ether (Brij-35) was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism (CD) spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses.

Sortase-catalyzed initiator attachment enables high yieldgrowth of a stealth polymer from the C terminus of a protein.

Conventional methods for synthesizing protein/peptide-polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site- specificity, which results in heterogeneous products with significantly compromised bioactivity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with >90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide-polymer conjugates.

The affinity purification and characterization of ATP synthase complexes from mitochondria

The mitochondrial F1-ATPase inhibitor protein, IF1, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF1 occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF1 with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1–60 of bovine IF1 with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

Engineering fluorescent protein substrates for the AAA+ Lon protease

AAA+ proteases, such as Escherichia coli Lon, recognize protein substrates by binding to specific peptide degrons and then unfold and translocate the protein into an internal degradation chamber for proteolysis. For some AAA+ proteases, attaching specific degrons to the N- or C-terminus of green fluorescent protein (GFP) generates useful substrates, whose unfolding and degradation can be monitored by loss of fluorescence, but Lon fails to degrade appropriately tagged GFP variants at a significant rate. Here, we demonstrate that Lon catalyzes robust unfolding and degradation of circularly permuted variants of GFP with a β20 degron appended to the N terminus or a sul20 degron appended to the C terminus. Lon degradation of non-permuted GFP-sul20 is very slow, in part because the enzyme cannot efficiently extract the degron-proximal C-terminal β-strand to initiate denaturation. The circularly permuted GFP substrates described here allow convenient high-throughput assays of the kinetics of Lon degradation in vitro and also permit assays of Lon proteolysis in vivo.

AtAPY1 and AtAPY2 Function as Golgi-Localized Nucleoside Diphosphatases in Arabidopsis thaliana

Nucleoside triphosphate diphosphohydrolases (NTPDases; apyrases) (EC 3.6.1.5) hydrolyze di- and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad divalent cation (Mg(2+), Ca(2+)) requirement for activation and are insensitive to inhibitors of F-type, P-type and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescent protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgi-localized GDA1 mutant, rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of the wild type show higher substrate preferences toward UDP compared with other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNA interference (RNAi) technology repressor lines. Microsomes from wild-type plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP-tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity, confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi-silenced lines all have an increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together, these results reveal that AtAPY1 and AtAPY2 are Golgi-localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.

Topogenesis of heterologously expressed fragments of the human Y4 GPCR

Fragments of large membrane proteins have the potential to facilitate structural analysis by NMR, but their folding state remains a concern. Here we determined the quality of folding upon heterologous expression for a series of N- or C-terminally truncated fragments of the human Y4 G-protein coupled receptor, amounting to six different complementation pairs. As the individual fragments lack a specific function that could be used to ascertain proper folding, we instead assessed folding on a basic level by studying their membrane topology and by comparing it to well-established structural models of GPCRs. The topology of the fragments was determined using a reporter assay based on C-terminal green fluorescent protein- or alkaline phosphatase-fusions. N-terminal fusions to Lep or Mistic were used if a periplasmic orientation of the N-terminus of the fragments was expected based on predictions. Fragments fused to Mistic expressed at comparably high levels, whereas Lep fusions were produced to a much lower extent. Though none of the fragments exclusively adopted one orientation, often the correct topology predominated. In addition, systematic analysis of the fragment series suggested that the C-terminal half of the Y4 receptor is more important for adopting the correct topology than the N-terminal part. Using the detergent dodecylphosphocholine, selected fragments were solubilized from the membrane and proved sufficiently stable to allow purification. Finally, as a first step toward reconstituting a functional receptor from two fragments, we observed a physical interaction between complementing fragments pairs upon co-expression.

Heat Shock Protein 90 Is Required for Conidiation and Cell Wall Integrity in Aspergillus fumigatus

Heat shock protein 90 (Hsp90) is a eukaryotic molecular chaperone. Its involvement in the resistance of Candida albicans to azole and echinocandin antifungals is well established. However, little is known about Hsp90's function in the filamentous fungal pathogen Aspergillus fumigatus. We investigated the role of Hsp90 in A. fumigatus by genetic repression and examined its cellular localization under various stress conditions. Failure to generate a deletion strain of hsp90 suggested that it is essential. Genetic repression of Hsp90 was achieved by an inducible nitrogen-dependent promoter (pniiA-Hsp90) and led to decreased spore viability, decreased hyphal growth, and severe defects in germination and conidiation concomitant with the downregulation of the conidiation-specific transcription factors brlA, wetA, and abaA. Hsp90 repression potentiated the effect of cell wall inhibitors affecting the β-glucan structure of the cell wall (caspofungin, Congo red) and of the calcineurin inhibitor FK506, supporting a role in regulating cell wall integrity pathways. Moreover, compromising Hsp90 abolished the paradoxical effect of caspofungin. Pharmacological inhibition of Hsp90 by geldanamycin and its derivatives (17-AAG and 17-DMAG) resulted in similar effects. C-terminal green fluorescent protein (GFP) tagging of Hsp90 revealed mainly cytosolic distribution under standard growth conditions. However, treatment with caspofungin resulted in Hsp90 accumulation at the cell wall and at sites of septum formation, further highlighting its role in cell wall stress compensatory mechanisms. Targeting Hsp90 with fungal-specific inhibitors to cripple stress response compensatory pathways represents an attractive new antifungal strategy.