A hierarchical model of accelerating factors to promote urban renewal and reconstruction of unsafe and old buildings.

The issue of urban renewal is complex and multifaceted. In this study, six specialists in the construction industry were invited to conduct audio interviews, which were compiled into verbatim text. The key phrases were extracted by Grounded theory, and three levels of coding were retrieved. The data were categorized into ten accelerating urban renewal factors in three constructs to establish an Analytic Hierarchy Process framework. Using institutional theory to construct outcomes based on grounded theory, transforming these into specific urban renewal relation issue elements. 113 AHP questionnaires were collected from five types of specialists, including practitioners, professionals, participants in urban renewal, academics, and government staff. The results show that relaxing the plot ratio control and incentives is ranked No. 1 by practitioners, participants in urban renewal, professionals, and academics, indicating that the factor is highly valued by specialists but neglected by government staff. Secondly, practitioners, academics, participants in urban renewal, and professionals identified incentives and rewards for urban renewal and enhancing the trust and credibility of urban renewal projects as crucial factors. However, the government staff showed a different weighting. This indicates that government staff is determined to accelerate urban renewal. Finally, the suggestion of this study is in line with the views of the specialists interviewed, who suggest that the government should hold public hearings regularly and seriously to listen to people and specialists. Only through public hearings can all parties reach a consensus. The government should consolidate the views of all parties to amend or enact a bill on urban renewal that is more in line with the changes of the times, including appropriately relaxing the control of building plot ratio and other accelerating factors, to promote urban renewal in Taiwan.

Dynamic expression of cathepsin L in the black soldier fly (Hermetia illucens) gut during Escherichia coli challenge.

The black soldier fly (BSF), Hermetia illucens, has the potential to serve as a valuable resource for waste bioconversion due to the ability of the larvae to thrive in a microbial-rich environment. Being an ecological decomposer, the survival of BSF larvae (BSFL) relies on developing an efficient defense system. Cathepsin L (CTSL) is a cysteine protease that plays roles in physiological and pathological processes. In this study, the full-length of CTSL was obtained from BSF. The 1,020-bp open reading frame encoded a preprotein of 339 amino acids with a predicted molecular weight of 32 kDa. The pro-domain contained the conserved ERFNIN, GNYD, and GCNGG motifs, which are all characteristic of CTSL. Homology revealed that the deduced amino acid sequence of BSF CTSL shared 74.22-72.99% identity with Diptera flies. Immunohistochemical (IHC) analysis showed the CTSL was predominantly localized in the gut, especially in the midgut. The mRNA expression of CTSL in different larval stages was analyzed by quantitative real-time PCR (RT-qPCR), which revealed that CTSL was expressed in the second to sixth instar, with the highest expression in the fifth instar. Following an immune challenge in vivo using Escherichia coli (E. coli), CTSL mRNA was significantly up-regulated at 6 h post-stimulation. The Z-Phe-Arg-AMC was gradually cleaved by the BSFL extract after 3 h post-stimulation. These results shed light on the potential role of CTSL in the defense mechanism that helps BSFL to survive against pathogens in a microbial-rich environment.

Exploration of the Catalytic Cycle Dynamics of Vigna Radiata H+-Translocating Pyrophosphatases Through Hydrogen-Deuterium Exchange Mass Spectrometry.

Vigna radiata H+-translocating pyrophosphatases (VrH+-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H+ into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H+-PPases has been elucidated, the H+ translocation mechanism of H+-PPases in the solution state remains unclear. In this study, we used hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H+-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H+-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H+-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston.

Structural basis for calcium-stimulating pore formation of Vibrio α-hemolysin.

Vibrio α-hemolysins (αHLs) are ß-pore-forming toxins secreted by Vibrio pathogens, crucial for the facilitation of bacterial infections through host cell lysis. These toxins are produced as inactive precursors, requiring proteolytic maturation and membrane association for activation within host tissues. Here, we investigate Vibrio campbellii αHL (VcαHL), and establish that its hemolytic activity is significantly stimulated by calcium ions, with an EC50 that aligns with physiological calcium concentrations. Furthermore, we illustrate the vital contribution of calcium ions to the oligomerization of VcαHL on membranes. Using X-ray crystallography and cryo-electron microscopy, we decipher both the immature and assembled structures of VcαHL and elucidate the conformational changes corresponding to toxin assembly. We also identify a calcium-binding module that is integral for VcαHL's calcium-dependent activation. These findings provide insights into the regulatory mechanisms of VcαHL and have the potential to inform the development of targeted therapeutic strategies against Vibrio infections.

MetaMOPE: a web service for mobile phase determination and fast chromatography peaks evaluation for metabolomics.

Motivation: Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used in metabolomics studies, while HILIC LC-MS is particularly suited for polar metabolites. Determining an optimized mobile phase and developing a proper liquid chromatography method tend to be laborious, time-consuming and empirical. Results: We developed a containerized web tool providing a workflow to quickly determine the optimized mobile phase by batch-evaluating chromatography peaks for metabolomics LC-MS studies. A mass chromatographic quality value, an asymmetric factor, and the local maximum intensity of the extracted ion chromatogram were calculated to determine the number of peaks and peak retention time. The optimal mobile phase can be quickly determined by selecting the mobile phase that produces the largest number of resolved peaks. Moreover, the workflow enables one to automatically process the repeats by evaluating chromatography peaks and determining the retention time of large standards. This workflow was validated with 20 chemical standards and successfully constructed a reference library of 571 metabolites for the HILIC LC-MS platform. Availability and implementation: MetaMOPE is freely available at https://metamope.cmdm.tw. Source code and installation instructions are available on GitHub: https://github.com/CMDM-Lab/MetaMOPE. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Crystal structures of dimeric and heptameric mtHsp60 reveal the mechanism of chaperonin inactivation.

Mitochondrial Hsp60 (mtHsp60) plays a crucial role in maintaining the proper folding of proteins in the mitochondria. mtHsp60 self-assembles into a ring-shaped heptamer, which can further form a double-ring tetradecamer in the presence of ATP and mtHsp10. However, mtHsp60 tends to dissociate in vitro, unlike its prokaryotic homologue, GroEL. The molecular structure of dissociated mtHsp60 and the mechanism behind its dissociation remain unclear. In this study, we demonstrated that Epinephelus coioides mtHsp60 (EcHsp60) can form a dimeric structure with inactive ATPase activity. The crystal structure of this dimer reveals symmetrical subunit interactions and a rearranged equatorial domain. The α4 helix of each subunit extends and interacts with its adjacent subunit, leading to the disruption of the ATP-binding pocket. Furthermore, an RLK motif in the apical domain contributes to stabilizing the dimeric complex. These structural and biochemical findings provide new insights into the conformational transitions and functional regulation of this ancient chaperonin.

scDrug: From single-cell RNA-seq to drug response prediction.

Single-cell RNA sequencing (scRNA-seq) technology allows massively parallel characterization of thousands of cells at the transcriptome level. scRNA-seq is emerging as an important tool to investigate the cellular components and their interactions in the tumor microenvironment. scRNA-seq is also used to reveal the association between tumor microenvironmental patterns and clinical outcomes and to dissect cell-specific effects of drug treatment in complex tissues. Recent advances in scRNA-seq have driven the discovery of biomarkers in diseases and therapeutic targets. Although methods for prediction of drug response using gene expression of scRNA-seq data have been proposed, an integrated tool from scRNA-seq analysis to drug discovery is required. We present scDrug as a bioinformatics workflow that includes a one-step pipeline to generate cell clustering for scRNA-seq data and two methods to predict drug treatments. The scDrug pipeline consists of three main modules: scRNA-seq analysis for identification of tumor cell subpopulations, functional annotation of cellular subclusters, and prediction of drug responses. scDrug enables the exploration of scRNA-seq data readily and facilitates the drug repurposing process. scDrug is freely available on GitHub at https://github.com/ailabstw/scDrug.

To join the rebuild or not? An exploration of the factors influencing the public's intention to participate in urban renewal.

Due to the lack of trust in the builder and indeterminate benefits, it is a struggle for people in Taiwan to make up their minds to participate in urban renewal. This leads to the completion rate of urban renewal of fewer than one ten-thousandth of the new construction needed. This study investigated the perspective on the research variables for people in Taiwan and how those influence their intention to participate in urban renewal. Using the Theory of Planned Behavior, the research framework is designed with the trust of urban renewal project builders and the perceived benefits of public participation as the independent variables. Attitudes, subjective norms, and perceived behavioral control are the mediating variables, and the general public's intention to participate in urban renewal is the dependent variable. A total of 545 valid questionnaires were collected through the survey. The results showed that the respondents' trust in the builder of the urban renewal project positively and significantly influenced their perceived benefits of the project, and the respondents' trust in the builder significantly influenced their subjective norms. The perceived benefits positively and significantly affected their attitudes and subjective norms, and people's attitudes, subjective norms, and perceived behavioral control positively and significantly affected their intention to participate in urban renewal. People's perceived benefits in urban renewal projects affected their participation intention through attitudes and subjective norms. The variable perceived benefits most strongly influenced people's intention to participate in urban renewal in this study. This study provides practical suggestions for the government and builders to increase people's intention to participate in urban renewal. This study modeled two independent variables, trust in the builder and perceived benefits, under the urban renewal context in Taiwan. In future works, other factors could be included, such as tax incentives, floor area rewards, and fair appraisal.

Transport efficiency of AtGTR1 dependents on the hydrophobicity of transported glucosinolates.

Glucosinolates (GLSs) are a group of secondary metabolites that are involved in the defense of herbivores. In Arabidopsis thaliana, Glucosinolate Transporter 1 (AtGTR1) transports GLSs with high affinity via a proton gradient-driven process. In addition to transporting GLSs, AtGTR1 also transports phytohormones, jasmonic acid-isoleucine (JA-Ile), and gibberellin (GA). However, little is known about the mechanisms underlying the broad substrate specificity of AtGTR1. Here, we characterized the substrate preference of AtGTR1 by using a yeast uptake assay, and the results revealed that GLS transport rates are negatively correlated with the hydrophobicity of substrates. Interestingly, the AtGTR1 showed a higher substrate affinity for GLSs with higher hydrophobicity, suggesting a hydrophobic substrate binding pocket. In addition, competition assays revealed that JA, salicylic acid (SA), and indole-3-acetic acid (IAA) competed with GLS for transport in yeast, suggesting a potential interaction of AtGTR1 with these phytohormones. To further characterize the functional properties of AtGTR1, mutagenesis experiments confirmed that the conserved EXXEK motif and Arg166 are essential for the GLS transport function. In addition, the purified AtGTR1 adopts a homodimeric conformation, which is possibly regulated by phosphorylation on Thr105. The phosphomimetic mutation, T105D, reduced its protein expression and completely abrogated its GLS transport function, indicating the essential role of phosphorylation on AtGTR1. In summary, this study investigated various factors associated with the GLS transport and increased our knowledge on the substrate preferences of AtGTR1. These findings contribute to understanding how the distribution of defense GLSs is regulated in plants and could be used to improve crop quality in agriculture.

Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition.

Leptospirosis is an overlooked zoonotic disease caused by pathogenic Leptospira depended on virulence of Leptospira and the host-pathogen interaction. Kidney is the major organ infected by Leptospira which causes tubulointerstitial nephritis. Leptospira outer membrane contains several virulence factors and an outer membrane protein A (OmpA) like protein (Loa22) is essential for virulence. Pull-down assays suggested that Loa22 was a potential Toll-Like Receptor 2 (TLR2) binding candidates from pathogenic Leptospira. Confocal microscopy was employed to observe the co-localization of TLR2 and Loa22-LPGN (Leptospira peptidoglycan) complexes. Atomic force microscopy (AFM), side-directed mutagenesis, and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the affinity between rLoa22, LPGN, and TLR2. Real time PCR was applied to measure the cytokines expression. Downstream signal transduction components were verified by western blot to evaluate the gene regulations. Mutation of two Loa22 key residues (Asp122 and Arg143) attenuated the affinities for LPGN. rLoa22-LPGN complexes were observed to co-localize with TLR2 and provoked inflammatory responses including CXCL8/IL8, hCCL2/MCP-1, and hTNF-α. Affinity studies suggested that Loa22-LPGN complexes elevated the affinity to TLR2 as compared to Loa22 protein. Downstream signals from TLR2 including p38, ERK, and JNK were regulated under rLoa22-LPGN complexes treatments. This study identified LPGN mediates interactions between Loa22 and TLR2 and induces downstream signals to trigger inflammatory responses. rLoa22-LPGN-TLR2 complexes reveal a novel binding mechanism for the innate immune system.

Overexpression of a multifunctional ß-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter.

The ß-glucosidase, which hydrolyzes the ß(1-4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of ß-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including ß-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2-4. We overexpressed the ß-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with ß-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of ß-glucosidase in plants could further increase. The plant-expressed ß-glucosidase had optimal activity at 80 °C and pH 5-6. In addition, the plant-expressed ß-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with ß-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial ß-glucosidase gene as a reporter in plants based on alternative ß-galactosidase activity.

Dual-focal-plane augmented reality head-up display using a single picture generation unit and a single freeform mirror.

Two or more focal planes are required in augmented reality head-up displays (AR HUDs) to respectively present basic and interactive driving information to car drivers; whereas, solutions using two separated picture generation units (PGUs) with two sets of optics incur increased cost, reduced reliability, and expanded volume. To develop an AR HUD using a single PGU and a single curved mirror, we propose to set two logically separated regions on a single PGU and optically relay one of them to a new position to create two focal planes. A single freeform mirror acquired through careful optical and mechanical design optimization produces high image quality in an eyebox of 120 mm by 60 mm, simultaneously for a far image (9 m, 10° by 3°) and a near image (2.5 m, 6° by 2°). Finally, an AR HUD product with a compact volume of 8.5 L is fabricated to experimentally verify the design.

Potassium Stimulation of IAA Transport Mediated by the Arabidopsis Importer AUX1 Investigated in a Heterologous Yeast System.

Auxin regulates diverse processes involved in plant growth and development. AUX1 is the first identified and most widely investigated auxin importer, and plays an important role in root gravitropism and the development of lateral root and root hair. However, the regulation of auxin transport by AUX1 is still not well understood. In this study, we examined the effect of metal ions on AUX1 transport function and found that the activity could be specifically stimulated four times by K+. Further experiments revealed the preference of KF on the enhancement of transport activity of AUX1 over KCl, KBr, and KI. In addition, the interaction between K+ and AUX1 confers AUX1 more resistant to thermal stress but more vulnerable to proteolysis. Conventional chemical modification indicated that the extracellular acidic amino acids of AUX1 play a key role in the K+ stimulation. Site-specific mutagenesis showed that the replacement of Asp166, Asp293, and Asp312 of AUX1 to alanine deteriorated the K+-stimulated auxin transport. By contrast, when these residues were mutated to glutamate, lysine, or asparagine, only the D312E variant restored the IAA transport activity to the wild-type level. It is thus convinced that D312 is presumably the most promising residue for the K+ stimulation on AUX1.

Self-Selecting Resistive Switching Scheme Using TiO2 Nanorod Arrays.

In this study, the resistive switching scheme using TiO2 nanorod arrays synthesized by a large-scale and low-cost hydrothermal process was reported. Especially, the nonlinear I-V characteristics of TiO2 nanorod arrays with a nonlinearity of up to ~10, which suppress the leakage current less than 10-4 Acm-2, were demonstrated, exhibiting a self-selecting resistive switching behavior. It provides a simple pathway for integration of RRAM crossbar arrays without additional stacking of active devices. The mechanisms of the nonlinear resistive switching behaviors were discussed in detail. In addition, the maximum array numbers of 79 for self-selecting RRAM cells were estimated. The results demonstrate an opportunity of using the concept of self-selecting resistive switching characteristics in a single material, which offers a new strategy to tackle the sneak path issue of RRAM in the crossbar arrays structure.

Resistive switching of Sn-doped In2O3/HfO2 core-shell nanowire: geometry architecture engineering for nonvolatile memory.

Core-shell NWs offer an innovative approach to achieve nanoscale metal-insulator-metal (MIM) heterostructures along the wire radial direction, realizing three-dimensional geometry architecture rather than planar type thin film devices. This work demonstrated the tunable resistive switching characteristics of ITO/HfO2 core-shell nanowires with controllable shell thicknesses by the atomic layer deposition (ALD) process for the first time. Compared to planar HfO2 thin film device configuration, ITO/HfO2 core-shell nanowire shows a prominent resistive memory behavior, including lower power consumption with a smaller SET voltage of ∼0.6 V and better switching voltage uniformity with variations (standard deviation(σ)/mean value (µ)) of VSET and VRESET from 0.38 to 0.14 and from 0.33 to 0.05 for ITO/HfO2 core-shell nanowire and planar HfO2 thin film, respectively. In addition, endurance over 103 cycles resulting from the local electric field enhancement can be achieved, which is attributed to geometry architecture engineering. The concept of geometry architecture engineering provides a promising strategy to modify the electric-field distribution for solving the non-uniformity issue of future RRAM.

Scalable graphene synthesised by plasma-assisted selective reaction on silicon carbide for device applications.

Graphene, a two-dimensional material with honeycomb arrays of carbon atoms, has shown outstanding physical properties that make it a promising candidate material for a variety of electronic applications. To date, several issues related to the material synthesis and device fabrication need to be overcome. Despite the fact that large-area graphene films synthesised by chemical vapour deposition (CVD) can be grown with relatively few defects, the required transfer process creates wrinkles and polymer residues that greatly reduce its performance in device applications. Graphene synthesised on silicon carbide (SiC) has shown outstanding mobility and has been successfully used to develop ultra-high frequency transistors; however, this fabrication method is limited due to the use of costly ultra-high vacuum (UHV) equipment that can reach temperatures over 1500 °C. Here, we show a simple and novel approach to synthesise graphene on SiC substrates that greatly reduces the temperature and vacuum requirements and allows the use of equipment commonly used in the semiconductor processing industry. In this work, we used plasma treatment followed by annealing in order to obtain large-scale graphene films from bulk SiC. After exposure to N2 plasma, the annealing process promotes the reaction of nitrogen ions with Si and the simultaneous condensation of C on the surface of SiC. Eventually, a uniform, large-scale, n-type graphene film with remarkable transport behaviour on the SiC wafer is achieved. Furthermore, graphene field effect transistors (FETs) with high carrier mobilities on SiC were also demonstrated in this study.

Tunable multilevel storage of complementary resistive switching on single-step formation of ZnO/ZnWO(x) bilayer structure via interfacial engineering.

Tunable multilevel storage of complementary resistive switching (CRS) on single-step formation of ZnO/ZnWOx bilayer structure via interfacial engineering was demonstrated for the first time. In addition, the performance of the ZnO/ZnWOx-based CRS device with the voltage- and current-sweep modes was demonstrated and investigated in detail. The resistance switching behaviors of the ZnO/ZnWOx bilayer ReRAM with adjustable RESET-stop voltages was explained using an electrochemical redox reaction model whose electron-hopping activation energies of 28, 40, and 133 meV can be obtained from Arrhenius equation at RESET-stop voltages of 1.0, 1.3, and 1.5 V, respectively. In the case of the voltage-sweep operation on the ZnO-based CRS device, the maximum array numbers (N) of 9, 15, and 31 at RESET-stop voltages of 1.4, 1.5, and 1.6 V were estimated, while the maximum array numbers increase into 47, 63, and 105 at RESET-stop voltages of 2.0, 2.2, and 2.4 V, operated by the current-sweep mode, respectively. In addition, the endurance tests show a very stable multilevel operation at each RESET-stop voltage under the current-sweep mode.

Large scale and orientation-controllable nanotip structures on CuInS2, Cu(In,Ga)S2, CuInSe2, and Cu(In,Ga)Se2 by low energy ion beam bombardment process: growth and characterization.

One-step facile methodology to create nanotip arrays on chalcopyrite materials (such as CuInS2, Cu(In,Ga)S2, CuInSe2, and Cu(In,Ga)Se2) via a low energy ion beam bombardment process has been demonstrated. The mechanism of formation for nanotip arrays has been proposed by sputtering yields of metals and reduction of metals induced by the ion beam bombardment process. The optical reflectance of these chalcopyrite nanotip arrays has been characterized by UV-vis spectrophotometer and the efficient light-trapping effect has been observed. Large scale (∼4'') and high density (10(10) tips/cm(2)) of chalcopyrite nanotip arrays have been obtained by using low ion energy (< 1 kV), short processing duration (< 30 min), and template-free. Besides, orientation and length of these chalcopyrite nanotip arrays are controllable. Our results can be the guide for other nanostructured materials fabrication by ion sputtering and are available for industrial production as well.

Functional and fluorescence analyses of tryptophan residues in H+-pyrophosphatase of Clostridium tetani.

Homodimeric proton-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PPi) hydrolysis and proton translocation. H+-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PPi binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H+-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-Pi binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H+-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H+-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis.

Functional investigation of transmembrane helix 3 in H⁺-translocating pyrophosphatase.

H⁺-translocating pyrophosphatase (H⁺-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PP(i)) hydrolysis. Vigna radiata H⁺-PPase (VrH⁺-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H⁺-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H⁺-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH⁺-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K⁺-mediated stimulation of H⁺-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PP(i) hydrolysis, proton transport, expression, and K⁺ stimulation of H⁺-PPase.