Probing the local secondary structure of bacteriophage S21 pinholin membrane protein using electron spin echo envelope modulation spectroscopy

There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S²¹ pinholin. This ESEEM approach uses a combination of site-directed spin labeling and ²H-labeled side chains. Pinholin S²¹ is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S²¹ pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S²¹ pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 3₁₀-helix which have been observed in other channel forming proteins.     

Mutational analysis of the S21 pinholin

Lambdoid phage 21 has the prototype pinholin‐SAR endolysin lysis system, which is widely distributed among phages. Its prototype pinholin, S²¹68, triggers at an allele‐specific time to form small, heptameric lesions, or pinholes, in the cytoplasmic membrane, thus initiating lysis. S²¹68 has two transmembrane domains, TMD1 and TMD2. Only TMD2 is required for the formation of pinholes, whereas TMD1 acts as an inhibitor of TMD2 and must be externalized to the periplasm in the lytic pathway. Previously we provided evidence that S²¹68 first accumulates as inactive dimers with both transmembrane domains embedded in the bilayer. Here we analyse an extensive collection of S²¹ mutants to identify residues and domains critical to the function and regulation of the pinholin. Evidence is presented indicating that, within the inactive dimer, TMD1 acts in trans as an inhibitor of the lethal function of TMD2. A wide range of phenotypes, from absolute lysis defectives to accelerated lysis triggering, are observed for mutations mapping to each topological domain. The pattern of phenotypes allows the generation of a model for the structure of the inactive dimer. The model identifies the faces of the two transmembrane domains involved in intramolecular and intermolecular interactions, as well as interaction with the lipid.     

Pinholin S21 mutations induce structural topology and conformational changes

The bacteriophage infection cycle is terminated at a predefined time to release the progeny virions via a robust lytic system composed of holin, endolysin, and spanin proteins. Holin is the timekeeper of this process. Pinholin S²¹ is a prototype holin of phage Φ21, which determines the timing of host cell lysis through the coordinated efforts of pinholin and antipinholin. However, mutations in pinholin and antipinholin play a significant role in modulating the timing of lysis depending on adverse or favorable growth conditions. Earlier studies have shown that single point mutations of pinholin S²¹ alter the cell lysis timing, a proxy for pinholin function as lysis is also dependent on other lytic proteins. In this study, continuous wave electron paramagnetic resonance (CW-EPR) power saturation and double electron-electron resonance (DEER) spectroscopic techniques were used to directly probe the effects of mutations on the structure and conformational changes of pinholin S²¹ that correlate with pinholin function. DEER and CW-EPR power saturation data clearly demonstrate that increased hydrophilicity induced by residue mutations accelerate the externalization of antipinholin transmembrane domain 1 (TMD1), while increased hydrophobicity prevents the externalization of TMD1. This altered hydrophobicity is potentially accelerating or delaying the activation of pinholin S²¹. It was also found that mutations can influence intra- or intermolecular interactions in this system, which contribute to the activation of pinholin and modulate the cell lysis timing. This could be a novel approach to analyze the mutational effects on other holin systems, as well as any other membrane protein in which mutation directly leads to structural and conformational changes.     

Display of active beta-glucosidase on the surface of Schizosaccharomyces pombe cells using novel anchor proteins

Here, we demonstrate display of beta-glucosidase (BGL) on the surface of Schizosaccharomyces pombe cells using novel anchor proteins. A total of four candidate anchor proteins (SPBC21D10.06c, SPBC947.04, SPBC19C7.05, and SPBC359.04c) were selected from among almost all of S. pombe membrane proteins. The C-terminus of each anchor protein was genetically fused to the N-terminus of BGL, and the fusion protein was expressed using S. pombe as a host. The highest cell surface-associated BGL activity (107 U/10⁵ cells was achieved with SPBC359.04c serving as the anchor, followed by SPBC947.04 (44 U/10⁵ cells) and SPBC21D10.06c (38 U/10⁵ cells). S. pombe displaying BGL with SPBC359.04c as an anchor showed the highest growth on 2 % cellobiose (10.7?×?10⁷ cells/mL after 41 h of cultivation from an initial density of 0.1?×?10⁷ cells/mL). Additionally, culturing BGL-displaying S. pombe in medium containing cellobiose as the sole carbon source did not affect protein expression, and ethanol fermentation from cellobiose was successfully demonstrated using BGL-displaying S. pombe. This is the first report describing a cell surface display system for the functionalization of S. pombe.     

Determining the helical tilt angle and dynamic properties of the transmembrane domains of pinholin S2168 using mechanical alignment EPR spectroscopy

The lytic cycle of bacteriophage φ21 for the infected E. coli is initiated by pinholin S²¹, which determines the timing of host cell lysis through the function of pinholin (S²¹68) and antipinholin (S²¹71). The activity of pinholin or antipinholin directly depends on the function of two transmembrane domains (TMDs) within the membrane. For active pinholin, TMD1 externalizes and lies on the surface while TMD2 remains incorporated inside the membrane forming the lining of the small pinhole. In this study, spin labeled pinholin TMDs were incorporated separately into mechanically aligned POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) lipid bilayers and investigated with electron paramagnetic resonance (EPR) spectroscopy to determine the topology of both TMD1 and TMD2 with respect to the lipid bilayer; the TOAC (2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid) spin label was used here because it attaches to the backbone of a peptide and is very rigid. TMD2 was found to be nearly colinear with the bilayer normal (n) with a helical tilt angle of 16 ± 4° while TMD1 lies on or near the surface with a helical tilt angle of 84 ± 4°. The order parameters (~0.6 for both TMDs) obtained from our alignment study were reasonable, which indicates the samples incorporated inside the membrane were well aligned with respect to the magnetic field (B₀). The data obtained from this study supports previous findings on pinholin: TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S²¹68. In this study, the helical tilt angle of TMD1 was measured for the first time. For TMD2 our experimental data corroborates the findings of the previously reported helical tilt angle by the Ulrich group.     

Acylation of non-specific phospholipase C4 determines its function in plant response to phosphate deficiency

Non-specific phospholipase C (NPC) is involved in plant growth, development and stress responses. To elucidate the mechanism by which NPCs mediate cellular functions, here we show that NPC4 is S-acylated at the C terminus and that acylation determines its plasma membrane (PM) association and function. The acylation of NPC4 was detected using NPC4 isolated from Arabidopsis and reconstituted in vitro. The C-terminal Cys-533 was identified as the S-acylation residue, and the mutation of Cys-533 to Ala-533 in NPC4 (NPC4^C533A) led to the loss of S-acylation and membrane association of NPC4. The knockout of NPC4 impeded the phosphate deficiency-induced decrease of the phosphosphingolipid glycosyl inositol phosphoryl ceramide (GIPC), but introducing NPC4^C533A to npc4-1 failed to complement this defect, thereby supporting the hypothesis that the non-acylated NPC4^C533A fails to hydrolyze GIPC during phosphate deprivation. Moreover, NPC4^C533A failed to complement the primary root growth in npc4-1 under stress. In addition, NPC4 in Brassica napus was S-acylated and mutation of the S-acylating cysteine residue of BnaC01.NPC4 led to the loss of S-acylation and its membrane association. Together, our results reveal that S-acylation of NPC4 in the C terminus is conserved and required for its membrane association, phosphosphingolipid hydrolysis and function in plant stress responses.     

基于比较转录组学分析NaCl胁迫影响德尔卑沙门氏菌的耐渗机制

【目的】研究高渗胁迫条件下德尔卑沙门氏菌(Salmonella enterica subsp. enterica Derby, S. Derby)的转录组调控机制,分析差异表达基因(differentially expressed genes, DEGs)表达水平,探究在高渗胁迫影响下德尔卑沙门氏菌耐渗反应的相关代谢通路。【方法】通过高渗胁迫诱导德尔卑沙门氏菌的耐渗性,提取菌株的总RNA,去除rRNA,构建cDNA文库。利用转录组测序技术及生物学信息技术分析相关DEGs,并通过实时荧光定量PCR (real-time fluorescence quantitative PCR, qRT-PCR)进行验证。【结果】胁迫组德尔卑沙门氏菌通过转录组测序结果发现有3 950个DEGs,其中具有显著上调的基因21个,显著下调基因38个。涉及到细胞膜蛋白、氨基酸的代谢等相关基因上调,协助德尔卑沙门氏菌在高渗环境中存活。与此同时,胁迫组德尔卑沙门氏菌的糖转运系统(sugar transport system, PTS)、糖酵解过程以及抗氧化性相关基因表达显著下调,这是由于高渗环境菌体需要在体内储存大量糖类等物质,从而降低了糖原的消耗,进而导致细胞外膜的脂多糖合成受到抑制,降低了高渗胁迫下德尔卑沙门氏菌细胞膜表面的O抗原的合成。【结论】高渗环境诱导后显著提高了德尔卑沙门氏菌的耐渗性,其中Na⁺/H⁺逆向转运蛋以及谷氨酸的代谢通路发挥着重要的作用,为进一步了解以及更好地控制其在食品中的污染提供了理论依据。     

Evidence for ΔpH surface component (ΔpH) of proton motive force in ATP synthesis of mitochondria

Background

One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, ΔµH⁺, or by more localized membrane surface proton pathways, as interfacial pmf, ΔµH^S.

Methods

We measure ?pH^S in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface.

Results

In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast ?pH^S, supporting that ΔµH^S is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with ?pH^S of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and ?pH^S, but not with ?Ψ_E.

General Significance

Our data substantiate ?pH^S as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of ?pH^S and ?Ψ_E in mitochondrial bioenergetics are also presented.     

Membrane interactions and dynamics of a 21-mer cytotoxic peptide: A solid-state NMR study

We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation, ³¹P and ²H NMR experiments were performed on 21-mer peptide-containing bicelles. ³¹P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand, ²H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding. ¹⁵N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface. ¹⁵N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally, ¹³C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the ¹³CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation.     

Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin

Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin’s antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.     

The dynamics of the G protein-coupled neuropeptide Y2 receptor in monounsaturated membranes investigated by solid-state NMR spectroscopy

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors’ function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static ¹⁵N NMR spectra and quantitative determination of ¹H–¹³C order parameters through measurement of the ¹H–¹³C dipolar couplings of the CH, CH₂ and CH₃ groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (S_backbone = 0.59–0.67, S_CH2 = 0.41–0.51 and S_CH3 = 0.22) obtained in directly polarized ¹³C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.     

Thiol protection in membrane protein purifications: A study with phage holins

The lambda holin, or S105, is a small cytoplasmic membrane protein that controls the timing of host lysis. Using thiol-specific reagents, we determined that the single cysteine residue within S105 was heterogeneously modified during membrane extraction and subsequent immobilized metal ion chromatography. Here we describe the use of a specific and reversible thiol reagent, 2,2′-dithiodipyridine, to generate purified protein with its cysteine residues in the native thiol state. The 2,2′-dithiodipyridine protection protocol was also successfully used for another unrelated holin, S²¹68, and should be generally useful for the purification of membrane proteins.     

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (S ^a, S ^b, S ^c, S ^d). We previously reported the cDNA cloning of three of these alleles, namely S ^b, S ^c and S ^d. In this paper we report the cloning and DNA sequence analysis of the S ^a allele. The S^a-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (S^b, S^c, and S^d) and this similarity was lower than that observed among the S^b, S^c and S^d-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; S ^a (602 bp), S ^b (1083 bp), S ^c (221 bp) and S ^d (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the S ^a- and S ^b-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the S ^a-allele and 556 bp in the S ^b-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage. Received: 21 June 1999 / Accepted: 15 November 1999     

Structural divergence among cannabinoids influences membrane dynamics: A H Solid-State NMR analysis

Cannabinoids are compounds that can modulate neuronal functions and immune responses via their activity at the CB₁ receptor. We used ²H NMR order parameters and relaxation rate determination to delineate the behavior of magnetically aligned phospholipid bilayers in the presence of several structurally distinct cannabinoid ligands. THC (Δ⁹-Tetrahydrocannabinol) and WIN-55,212-2 were found to lower the phase transition temperature of the DMPC and to destabilize their acyl chains leading to a lower average S_CD (≈ 0.13), while methanandamide and CP-55,940 exhibited unusual properties within the lipid bilayer resulting in a greater average S_CD (≈ 0.14) at the top of the phospholipid upper chain. The CB₁ antagonist AM281 had average S_CD values that were higher than the pure DMPC lipids, indicating a stabilization of the lipid bilayer. R_1Z versus |S_CD|² plots indicated that the membrane fluidity is increased in the presence of THC and WIN-55,212-2. The interaction of CP-55,940 with a variety of zwitterionic and charged membranes was also assessed. The unusual effect of CP-55,940 was present only in bicelles composed of DMPC. These studies strongly suggest that cannabinoid action on the membrane depends upon membrane composition as well as the structure of the cannabinoid ligands.     

A new approach to the investigation on the tonogenic groups of root cell walls

Acid-base properties of wheat, lupin, pea root cell walls were investigated. The roots of etiolated and green plants of different age were analysed by the potentiometric method. The ion exchange capacity of root cell walls (S_i) was estimated at various pH values (pH_i 2 to pH_i 12) and constant ion strength of the solution (10 mM). To analyse polysigmoid curves pH_i =f (S_i), Gregor's equation was used. It was shown that Gregor's model fits fairly well the experimental data. The total quantities of cation-exchange (S_t ^cat) and anion-exchange (S_t ^an) groups were determined in the root cell walls. It was shown that the quantity of anion exchange groups is varied through a small range (60–185 μmol/g dry wt.) in plant species tested, and that the S_t ^cat differs widely from 550 to 1300 μmol/g dry wt. For leguininous plants the quantity of acidic groups (fixed anions) is nearly twice as large as that for cereals. It was found that in seedlings as well as in plants, there are 3 cation-exchange groups and one anion-exchange group in root cell walls. The quantity of functional groups of each type (S^j) was estimated, and the corresponding values of n^j and pK_a ^j were calculated. It can be assumed that the groups with the pK_a ¹ ≈ 3.2 are amine groups, the ones with PK_a ² ≈ 5 are groups of galacturonic acid, the ones with pK_a ≈ 7.5 are the carboxyl groups of the second species, and the ones with pK_a ⁴ ≈ 40 are the phenolic groups. The values of dissociation constants (pK_a ^j) and S^j indicate that the root cell walls of wheat, lupin and pea are identical in qualitative structure of ionogenic groups but vary in the quantity of each ionogenic group. It was demonstrated that the summarized quantity of carboxyl groups (S² + S³) should be connected directly with the pH gradient in the extracellar space at the membrane surface. The gradient arises from ion-exchange reactions between cations of an outer medium and protons of the ionized carboxyl groups of the cell walls. The results suggest that, S_t ^cat and S_t ^an allow the quantitative estimation of ion exchange properties of the cell walls. The resulting parameters (S^j, pK_a ^j and n^j) allow prediction of changes in an ionic composition of a medium that bathes the cell membrane, during the first step of mineral nutrition uptake. This revised version was published online in June 2006 with corrections to the Cover Date.     

Study on the properties of the S3-state by mass spectrometry in the filamentous cyanobacterium Oscillatoria chalybea

In the present paper we analyzed the properties of the S₃-state in the filamentous cyanobacterium Oscillatoria chalybea by mass spectrometry. In this organism a substantial O₂-signal due to a single flash is observed even after extensive dark adaptation (20 min). This signal can be measured by mass spectrometry as well as amperometrically on an oxygen electrode and is not due to an interference of respiratory and photosynthetic electron transport in the prokaryotic membrane. The mass spectrometric analysis shows that, if S₃ is generated by two flashes in a medium containing only H₂¹⁶O, addition of H₂¹⁸O and subsequent firing of a third flash yields O₂ evolution labelled with ¹⁸O. It appears that the isotopic composition of the O₂ evolved corresponds to the isotopic composition of the water in the suspension. This experiment shows that water oxidation does not proceed via an oxygen precursor or water derivatives bound to the S₃-state. This conclusion has been reached shortly before ours by Radmer and Ollinger [15] in the reverse marker experiment. From our study with O. chalybea it appears that freshly generated S₃ can be distinguished from metastable S₃ by the mass spectrometric method. It looks as if in contrast to freshly generated S₃ metastable S₃ contained bound unexchangeable water or an oxidized water derivative.     

Molecular basis of the optochin-sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniaeStreptococcus oralis H+-ATPases

The gene responsible for the optochin-sensitive (Opt^S) phenotype of Streptococcus pneumoniae has been characterized. Sequence comparisons indicated that the genes involved encoded the subunits of the F₀ complex of an H⁺-ATPase. Sequence analysis and transformation experiments showed that the atpC gene is responsible for the optochin-sensitive resistant (Opt^S/Opt^R) phenotype. Our results also show that natural as well as laboratory Opt^R isolates have arisen by point mutations that produce different amino acid changes at positions 48, 49 or 50 of the ATPase c subunit. The nucleotide sequence of the F F₀ complex of the Streptococcus oralis ATPase has also been determined. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (Opt^S) and M222 (an Opt^R strain produced by inter-species recombination between pneumococcus and S. oralis), and S. oralis revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin specifically inhibited the membrane-bound ATPase activity of the S. pneumoniae wild-type (Opt^S) strains, and found a 100-fold difference between Opt^S and Opt^R strains, both in growth inhibition and in membrane ATPase resistance.     

Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3 ⁺-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3 ⁺ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3 ⁺-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.