RESUMO
Cytochromes c'-α are nitric oxide (NO)-binding heme proteins derived from bacteria that can thrive in a wide range of temperature environments. Studies of mesophilic Alcaligenes xylosoxidans cytochrome c'-α (AxCP-α) have revealed an unusual NO-binding mechanism involving both heme faces, in which NO first binds to form a distal hexa-coordinate Fe(II)-NO (6cNO) intermediate and then displaces the proximal His to form a proximal penta-coordinate Fe(II)-NO (5cNO) final product. Here, we characterize a thermally stable cytochrome c'-α from thermophilic Hydrogenophilus thermoluteolus (PhCP-α) to understand how protein thermal stability affects NO binding. Electron paramagnetic and resonance Raman spectroscopies reveal the formation of a PhCP-α 5cNO product, with time-resolved (stopped-flow) UV-vis absorbance indicating the involvement of a 6cNO intermediate. Relative to AxCP-α, the rates of 6cNO and 5cNO formation in PhCP-α are â¼11- and â¼13-fold lower, respectively. Notably, x-ray crystal structures of PhCP-α in the presence and absence of NO suggest that the sluggish formation of the proximal 5cNO product results from conformational rigidity: the Arg-132 residue (adjacent to the proximal His ligand) is held in place by a salt bridge between Arg-75 and Glu-135 (an interaction not present in AxCP-α or a psychrophilic counterpart). Overall, our data provide fresh insights into structural factors controlling NO binding in heme proteins, including 5cNO complexes relevant to eukaryotic NO sensors.
RESUMO
The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-ß) resembling that of cytochromes P460. A recent structure of cyt c'-ß from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c'-ß but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c'-ß from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-ß, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.
Assuntos
Citocromos c' , Methylococcus capsulatus , Humanos , Citocromos c'/química , Gases , Heme/metabolismo , Hemeproteínas/genética , Hemeproteínas/metabolismo , Methylococcus capsulatus/químicaRESUMO
Two homologous cytochromes c', SBCP and SVCP, from deep-sea Shewanella benthica and Shewanella violacea respectively exhibit only nine surface amino acid substitutions, along with one at the N-terminus. Despite the small sequence difference, SBCP is thermally more stable than SVCP. Here, we examined the thermal stability of SBCP variants, each containing one of the nine substituted residues in SVCP, and found that the SBCP K87V variant was the most destabilized. We then determined the X-ray crystal structure of the SBCP K87V variant at a resolution of 2.1 Å. The variant retains a four-helix bundle structure similar to the wild-type, but notable differences are observed in the hydration structure around the mutation site. Instead of forming of the intrahelical salt bridge between Lys-87 and Asp-91 in the wild-type, a clathrate-like hydration around Val-87 through a hydrogen bond network with the nearby amino acid residues is observed. This network potentially enhances the ordering of surrounding water molecules, leading to an entropic destabilization of the protein. These results suggest that the unfavorable hydrophobic hydration environment around Val-87 and the inability to form the Asp-91-mediated salt bridge contribute to the observed difference in stability between SBCP and SVCP. These findings will be useful in future protein engineering for controlling protein stability through the manipulation of surface intrahelical salt bridges.
Assuntos
Citocromos c' , Citocromos c , Citocromos c/química , Citocromos c/genética , Citocromos c/metabolismo , Citocromos c'/metabolismo , Conformação Proteica , Estabilidade ProteicaRESUMO
AIMS: Certain lactic acid bacteria (LAB) are known to have anti-inflammatory effects; however, hiochi bacteria, which are taxonomically classified as LAB and known to spoil a traditional Japanese alcoholic beverage, have not been studied in the same context. The aim of this study is to investigate the anti-inflammatory effects of hiochi bacteria strains and the underlying mechanisms. METHODS AND RESULTS: We screened 45 strains of hiochi bacteria for anti-inflammatory effects and found that Lentilactobacillus hilgardii H-50 strongly inhibits lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in mouse splenocytes. This inhibition is attributed to its specific surface layer proteins (SLPs), which directly bind to LPS. CONCLUSIONS: The L. hilgardii H-50 strain exerts anti-inflammatory effects through its SLPs.
Assuntos
Lipopolissacarídeos , Baço , Camundongos , Animais , Lipopolissacarídeos/farmacologia , Baço/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anti-Inflamatórios/farmacologiaRESUMO
Candidatus Vesicomyosocius okutanii is a currently uncultured endosymbiotic bacterium of Phreagena okutanii, a clam that inhabits deep-sea vent environments. The genome of Ca. V. okutanii encodes a sulfur-oxidizing (Sox) enzyme complex, presumably generating biological energy for the host from inorganic sulfur compounds. Here, Ca. V. okutanii SoxX (VoSoxX), a mono-heme cytochrome c component of the Sox complex, was shown to be phylogenetically related to its homologous counterpart (HcSoxX) from a free-living deep-sea bacterium, Hydrogenovibrio crunogenus. Both proteins were heterologously expressed in Escherichia coli co-expressing cytochrome c maturation genes for comparative biochemical analysis. The VoSoxX recombinant had significantly lower thermal stability than HcSoxX, reflecting the difference in growth conditions of the source bacteria. The endosymbiont inhabits a mild intracellular environment, whereas the free-living bacterium dwells in a harsh environment. This study represents the first successful case of heterologous expression of genes from Ca. V. okutanii, allowing further biochemical studies of the molecular mechanism of sulfur oxidation in deep-sea environments.
Assuntos
Bivalves , Gammaproteobacteria , Animais , Bactérias/genética , Bivalves/genética , Bivalves/metabolismo , Citocromos c , Filogenia , Piscirickettsiaceae , Enxofre/metabolismo , Compostos de EnxofreRESUMO
Hydrogenophilus thermoluteolus, Thermochromatium tepidum, and Allochromatium vinosum, which grow optimally at 52, 49, and 25 °C, respectively, have homologous cytochromes c' (PHCP, TTCP, and AVCP, respectively) exhibiting at least 50% amino acid sequence identity. Here, the thermal stability of the recombinant TTCP protein was first confirmed to be between those of PHCP and AVCP. Structure comparison of the 3 proteins and a mutagenesis study on TTCP revealed that hydrogen bonds and hydrophobic interactions between the heme and amino acid residues were responsible for their stability differences. In addition, PHCP, TTCP, and AVCP and their variants with altered stability similarly bound nitric oxide and carbon oxide, but not oxygen. Therefore, the thermal stability of TTCP together with PHCP and AVCP can be tuned through specific interactions around the heme without affecting their gas-binding function. These cytochromes c' will be useful as specific gas sensor proteins exhibiting a wide thermal stability range.
Assuntos
Proteínas de Bactérias/metabolismo , Chromatiaceae/enzimologia , Citocromos c'/metabolismo , Gases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Chromatiaceae/crescimento & desenvolvimento , Dicroísmo Circular , Cristalografia por Raios X , Citocromos c'/química , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
An extract of date (fruit of a palm tree) residue plus food-grade glutamate, acetic acid, and yeast extract (date residue extract mix, DREM) has been successfully fermented with using Lactobacillus brevis JCM 1059T to produce gamma-aminobutyric acid (GABA). Here, mouse splenocytes were found to be viable when supplemented with DREM and fermented DREM containing GABA (fDREM). The addition of DREM and fDREM resulted in the secretion of tumor necrosis factor (TNF)-α from the splenocytes, fDREM being more effective than DREM. The TNF-α secretion with DREM was elevated by exogenous addition of GABA and that with fDREM was in part mediated via A-type GABA receptors. Contrary to general understanding of the suppressive effects of GABA on various biological functions, our findings suggest that GABA-containing fDREM arguments the immune function as a food and pharmaceutical material.
Assuntos
Cronologia como Assunto , Fermentação , Phoeniceae/química , Extratos Vegetais/química , Baço/citologia , Ácido gama-Aminobutírico/química , Animais , Feminino , Levilactobacillus brevis/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Gamma-aminobutyric acid (GABA) is produced by Lactobacillus brevis using date residue fermentation. In this study, the GABA production method was improved, for which L. brevis strain JCM 1059T was the most efficient among the four L. brevis strains examined. This was presumably due to a difference in the expression level of the gene encoding glutamate decarboxylase that catalyzes GABA synthesis.Abbreviation: GABA: gamma-aminobutyric acid.
Assuntos
Glutamato Descarboxilase/genética , Levilactobacillus brevis/enzimologia , Levilactobacillus brevis/genética , Phoeniceae/química , Extratos Vegetais/metabolismo , Ácido gama-Aminobutírico/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Glutamato Descarboxilase/metabolismo , Concentração de Íons de Hidrogênio , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neutrophilic Shewanella violacea is isolated from deep-sea sediments and its response to high pressure and high salinity has been investigated. Here, the pure effects of acidic pH on S. violacea physiology were examined, aiming at further understanding of its stress response mechanism. S. violacea could grow at initial pH of 5.0-7.0 without pH adjustment during the test at atmospheric pressure, and the lowest growth rate was obtained at pH 5.0. The pH of the same growth culture with an initial pH of 5.0 rose toward a neutral pH of ~ 7.0 at the exponential growth phase, indicating that S. violacea has a mechanism for acid neutralization. When S. violacea cells were grown at the fixed pH of 5.0, about five times higher concentrations of butyric and isovaleric acids were produced than at pH 7.0. The expression level of the genes encoding three enzymes for isovaleric acid synthesis from L-leucine was also found to be upregulated in S. violacea cells grown at the fixed pH of 5.0 compared with at pH 7.0 through RNA-seq analysis. Therefore, S. violacea at least produces isovaleric acid in its response to acid stress, which further deepens our understanding of the stress response mechanism inherent in this bacterium.
Assuntos
Ácido Butírico/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Ácidos Pentanoicos/metabolismo , Shewanella/metabolismo , Estresse Fisiológico/fisiologia , Hemiterpenos , Concentração de Íons de HidrogênioRESUMO
The stability of dimeric cytochrome c' from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the heme and at the subunit-subunit interface, both of which are molecular interior regions. Here, we showed that interactions in the equivalent interior regions of homologous cytochromes c' from two psychrophiles, Shewanella benthica and Shewanella violacea (SBCP and SVCP, respectively) were similarly weakened as compared with those of the counterparts of psychrophilic Shewanella livingstonensis and mesophilic Shewanella amazonensis (SLCP and SACP, respectively), and consistently the stability of SVCP, SLCP, and SACP increased in that order. Therefore, the stability of cytochromes c' from the psychrophile, mesophile, and thermophile is systematically regulated in their molecular interior regions. Unexpectedly, however, the stability of SBCP was significantly higher than that of SVCP, and the former had additional molecular surface interactions. Collectively, SBCP had weakened interior interactions like SVCP did, but the former was stabilized at the molecular surface as compared with the latter, implying complex multiple adaptation of the proteins because the psychrophilic sources of SBCP and SVCP are also piezophilic, thriving in deep-sea extreme environments of low temperature and high hydrostatic pressure.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Shewanella/metabolismo , Proteínas de Bactérias/química , Temperatura Baixa , Grupo dos Citocromos c/química , Estabilidade Enzimática , Pressão Hidrostática , Shewanella/genéticaRESUMO
Deep-sea Shewanella violacea 5'-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions. Abbreviations: NTase: 5'-nucleotidase; SANTase: Shewanella amazonensis 5'-nucleotidase; SVNTase: Shewanella violacea 5'-nucleotidase; CD: circular dichroism.
Assuntos
5'-Nucleotidase/metabolismo , Água do Mar/microbiologia , Shewanella/enzimologia , 5'-Nucleotidase/química , Adenosina Trifosfatases/metabolismo , Biocatálise , Domínio Catalítico , Dicroísmo Circular , Estabilidade Enzimática , Temperatura Alta , Compostos Inorgânicos/química , Compostos Orgânicos/química , Conformação Proteica , Tolerância ao Sal , Shewanella/fisiologia , Solventes/químicaRESUMO
AVCP cytochrome c' from mesophilic Allochromatium vinosum exhibits lower stability than a thermophilic counterpart, Hydrogenophilus thermoluteolus cytochrome c' (PHCP), in which the six specific amino acid residues that are not conserved in AVCP are responsible for its stability. Here we measured the stability of AVCP variants carrying these specific residues instead of the original AVCP ones. Among the six single AVCP variants, all of which formed a dimeric structure similar to that of the wild-type, three were successfully stabilized compared with the wild-type, while one showed lower stability than the wild-type. In addition, the most stabilized and destabilized AVCP variants could bind CO, similar to the wild-type. These results indicated that mesophilic AVCP could be stabilized through specific three mutations modeled by the thermophilic counterpart, PHCP, without changing the CO binding ability.
Assuntos
Chromatiaceae/enzimologia , Citocromos c/genética , Citocromos c/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Homologia de Sequência de Aminoácidos , Chromatiaceae/genética , Citocromos c/química , Estabilidade Enzimática , Modelos Moleculares , Proteínas Mutantes/química , Conformação Proteica , TemperaturaRESUMO
Two cytochromes c5 (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1-150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c551 (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.
RESUMO
The soluble protein fraction of the extremely halophilic archaeon Haloarcula japonica exhibits substantial inorganic pyrophosphate (PPi) hydrolysis activity in the presence of 2-4 M NaCl (Wakai et al, J Biol Chem 288:29247-29251, 2013), which provides high ionic strength (2-4). In this study, much higher PPi hydrolysis activity was unexpectedly detected, even with 0 M NaCl in the presence of 100-200 mM MgSO4, providing a much lower ionic strength of 0.4-0.8, in the same protein fraction. Na+ and Mg2+ ions were required for activity under high and low ionic strength conditions, respectively. A recombinant H. japonica pyrophosphatase (HjPPase) exhibited PPi hydrolysis activity with the same broad ionic strength range, indicating that the activity associated with such a broad ionic strength range could be attributed to a single enzyme. Thus, we concluded that the broad ionic strength range of HjPPase may contribute to adaptation for both Na+ and Mg2+ which are abundant but variable in the unstable living environments of H. japonica.
Assuntos
Proteínas Arqueais/metabolismo , Difosfatos/metabolismo , Haloarcula/enzimologia , Pirofosfatases/metabolismo , Proteínas Arqueais/química , Ambientes Extremos , Haloarcula/metabolismo , Concentração Osmolar , Pirofosfatases/química , SalinidadeRESUMO
Shewanella species are widely distributed in sea, brackish, and fresh water areas, growing psychrophilically or mesophilically, and piezophilically or piezo-sensitively. Here, membrane-bound 5'-nucleotidases (NTases) from deep-sea Shewanella violacea and brackish water Shewanella amazonensis were examined from the aspect of NaCl tolerance to gain an insight into protein stability against salt. Both NTases were single polypeptides with molecular masses of ~59 kDa, as determined on mass spectroscopy. They similarly required 10 mM MgCl2 for their activities, and they exhibited the same pH dependency and substrate specificity for 5'-nucleotides. However, S. violacea 5'-nucleotidase (SVNTase) was active enough in the presence of 2.5 M NaCl, whereas S. amazonensis 5'-nucleotidase (SANTase) exhibited significantly reduced activity with the same concentration of the salt. Although SVNTase and SANTase exhibited high sequence identity (69.7%), differences in the ratio of acidic to basic amino acid residues and the number of potential salt bridges maybe being responsible for the difference in the protein stability against salt. 5'-Nucleotidases from these Shewanella species will provide useful information regarding NaCl tolerance, which may be fundamental for understanding bacterial adaptation to growth environments.
Assuntos
5'-Nucleotidase/química , Proteínas de Bactérias/química , Membrana Celular/enzimologia , Água do Mar/microbiologia , Shewanella/enzimologia , Cloreto de Sódio/química , Microbiologia da Água , Shewanella/isolamento & purificaçãoRESUMO
Reversible denaturation of Pseudomonas aeruginosa cytochrome c551 (PAc551) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc551 denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc551 denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc551, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.
Assuntos
Proteínas de Bactérias/química , Grupo dos Citocromos c/química , Compostos de Metilureia/química , Pseudomonas aeruginosa/química , Ureia/análogos & derivados , Ureia/química , Proteínas de Bactérias/isolamento & purificação , Grupo dos Citocromos c/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Compostos de Metilureia/farmacologia , Desnaturação Proteica/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-Atividade , Termodinâmica , Ureia/farmacologiaRESUMO
Monomeric cytochrome c5 from deep-sea piezophilic Shewanella violacea (SVcytc5) was stable against heat and denaturant compared with the homologous protein from shallow-sea piezo-sensitive Shewanella livingstonensis (SLcytc5). Here, the SVcytc5 crystal structure revealed that the Lys-50 side chain on the flexible loop formed a hydrogen bond with heme whereas that of corresponding hydrophobic Leu-50 could not form such a bond in SLcytc5, which appeared to be one of possible factors responsible for the difference in stability between the two proteins. This structural insight was confirmed by a reciprocal mutagenesis study on the thermal stability of these two proteins. As SVcytc5 was isolated from a deep-sea piezophilic bacterium, the present comparative study indicates that adaptation of monomeric SVcytc5 to high pressure environments results in stabilization against heat.
Assuntos
Citocromos c/química , Shewanella/enzimologia , Cristalografia por Raios X , Citocromos c/genética , Citocromos c/metabolismo , Estabilidade Enzimática , Heme/química , Ligação de Hidrogênio , Modelos Moleculares , Mutagênese , Mutação , Conformação Proteica , TemperaturaRESUMO
Cytochrome c' (SACP) from mesophilic Shewanella amazonensis, growing optimally at 37 °C, was thermally more stable than cytochrome c' (AVCP) from mesophilic Allochromatium vinosum, growing optimally at 25 °C. In contrast, SACP was less stable than cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus, growing optimally at 52 °C. Although only 28% of the SACP amino acid sequence was identical to those of AVCP and PHCP, the latter two being 55% identical, the overall main chain structures of the three cytochromes c' were similar, and SACP exhibited thermal stability intermediate between those of AVCP and PHCP. For these three proteins, the higher the stability is, the lesser the number of Gly residues in the putative α-helical regions is. Cytochromes c' including the present three are suitable for examining the protein stabilization mechanisms, because they are structurally similar and available from environments with a wide range of temperatures.
Assuntos
Citocromos c/química , Shewanella/enzimologia , Sequência de Aminoácidos , Chromatiaceae/enzimologia , Dicroísmo Circular , Citocromos c/metabolismo , Estabilidade Enzimática , Hydrogenophilaceae/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , Homologia de Sequência de Aminoácidos , Shewanella/crescimento & desenvolvimento , Temperatura , TermodinâmicaRESUMO
Apo-cytochomes c without heme are usually unstructured. Here we showed that apo-form of thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) was a monomeric protein with high helix content. Apo-PHCP was thermally stable, possibly due to the hydrophobic residues and ion pairs. PHCP is the first example of a structured apo-cytochrome c', which will expand our view of hemoprotein structure formation.
Assuntos
Citocromos c/química , Hydrogenophilaceae/enzimologia , Sequência de Aminoácidos , Apoenzimas/química , Estabilidade Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus-vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.