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1.
Molecules ; 26(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34443607

RESUMO

Cellular conformation of reduced pyridine nucleotides NADH and NADPH sensed using autofluorescence spectroscopy is presented as a real-time metabolic indicator under pressurized conditions. The approach provides information on the role of pressure in energy metabolism and antioxidant defense with applications in agriculture and food technologies. Here, we use spectral phasor analysis on UV-excited autofluorescence from Saccharomyces cerevisiae (baker's yeast) to assess the involvement of one or multiple NADH- or NADPH-linked pathways based on the presence of two-component spectral behavior during a metabolic response. To demonstrate metabolic monitoring under pressure, we first present the autofluorescence response to cyanide (a respiratory inhibitor) at 32 MPa. Although ambient and high-pressure responses remain similar, pressure itself also induces a response that is consistent with a change in cellular redox state and ROS production. Next, as an example of an autofluorescence response altered by pressurization, we investigate the response to ethanol at ambient, 12 MPa, and 30 MPa pressure. Ethanol (another respiratory inhibitor) and cyanide induce similar responses at ambient pressure. The onset of non-two-component spectral behavior upon pressurization suggests a change in the mechanism of ethanol action. Overall, results point to new avenues of investigation in piezophysiology by providing a way of visualizing metabolism and mitochondrial function under pressurized conditions.


Assuntos
NADP/química , NADP/metabolismo , NAD/química , NAD/metabolismo , Pressão , Fluorescência , Conformação Molecular
2.
Microbiol Resour Announc ; 9(47)2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33214294

RESUMO

The draft genome sequence of the deep-sea yeast Naganishia liquefaciens strain N6, isolated from the Japan Trench, is reported here. This strain was previously classified into a Cryptococcus clade. Phylogenetic analysis using the presented sequence suggests that strain N6 is in the clade of the genus Naganishia.

3.
J Cell Sci ; 133(17)2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32801125

RESUMO

Mechanical stresses, including high hydrostatic pressure, elicit diverse physiological effects on organisms. Gtr1, Gtr2, Ego1 (also known as Meh1) and Ego3 (also known as Slm4), central regulators of the TOR complex 1 (TORC1) nutrient signaling pathway, are required for the growth of Saccharomyces cerevisiae cells under high pressure. Here, we showed that a pressure of 25 MPa (∼250 kg/cm2) stimulates TORC1 to promote phosphorylation of Sch9, which depends on the EGO complex (EGOC) and Pib2. Incubation of cells at this pressure aberrantly increased glutamine and alanine levels in the ego1Δ, gtr1Δ, tor1Δ and pib2Δ mutants, whereas the polysome profiles were unaffected. Moreover, we found that glutamine levels were reduced by combined deletions of EGO1, GTR1, TOR1 and PIB2 with GLN3 These results suggest that high pressure leads to the intracellular accumulation of amino acids. Subsequently, Pib2 loaded with glutamine stimulates the EGOC-TORC1 complex to inactivate Gln3, downregulating glutamine synthesis. Our findings illustrate the regulatory circuit that maintains intracellular amino acid homeostasis and suggest critical roles for the EGOC-TORC1 and Pib2-TORC1 complexes in the growth of yeast under high hydrostatic pressure.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Aminoácidos , Homeostase , Pressão Hidrostática , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Sci Rep ; 9(1): 18341, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31797992

RESUMO

Previously, we isolated 84 deletion mutants in Saccharomyces cerevisiae auxotrophic background that exhibited hypersensitive growth under high hydrostatic pressure and/or low temperature. Here, we observed that 24 deletion mutants were rescued by the introduction of four plasmids (LEU2, HIS3, LYS2, and URA3) together to grow at 25 MPa, thereby suggesting close links between the genes and nutrient uptake. Most of the highly ranked genes were poorly characterized, including MAY24/YPR153W. May24 appeared to be localized in the endoplasmic reticulum (ER) membrane. Therefore, we designated this gene as EHG (ER-associated high-pressure growth gene) 1. Deletion of EHG1 led to reduced nutrient transport rates and decreases in the nutrient permease levels at 25 MPa. These results suggest that Ehg1 is required for the stability and functionality of the permeases under high pressure. Ehg1 physically interacted with nutrient permeases Hip1, Bap2, and Fur4; however, alanine substitutions for Pro17, Phe19, and Pro20, which were highly conserved among Ehg1 homologues in various yeast species, eliminated interactions with the permeases as well as the high-pressure growth ability. By functioning as a novel chaperone that facilitated coping with high-pressure-induced perturbations, Ehg1 could exert a stabilizing effect on nutrient permeases when they are present in the ER.


Assuntos
Transporte Biológico/genética , Retículo Endoplasmático/genética , Proteínas de Membrana Transportadoras/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/genética , Retículo Endoplasmático/enzimologia , Proteínas de Membrana/genética , Membranas/enzimologia , Pressão , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética
5.
Biochem Biophys Res Commun ; 509(4): 1047-1052, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30660361

RESUMO

In Saccharomyces cerevisiae, high-affinity tryptophan import is mediated by the plasma membrane permease Tat2. Herein, we identified hyperactive Tat2 mutations, I285V and I285T, which allowed the cells to grow at very low tryptophan concentrations (<4 µg/mL). The Km value of wild-type Tat2 for tryptophan appeared to be 24 µg/mL, whereas that of Tat2I285V and Tat2I285T was 17 and 11 µg/mL, respectively. Normalized values of Vmax/Km for Tat2I285V- and Tat2I285T-mediated tryptophan import were 2-fold higher than that for Tat2, suggesting that these mutations increase the affinity for tryptophan, and mediate transport at very low tryptophan concentrations. I285 resides adjacent to E286, a fully conserved residue among amino acid pemreases. According to a pKa prediction for E208 (pKa ∼8.3-11.7) of Escherichia coli AdiC antiporter, a structural homologue of Tat2, the E286 carboxyl chain of Tat2 could get loaded with a proton during tryptophan/H+ symport. Hence, I285V and I285T mutations might affect the buried residue environment of Tat2, thereby facilitating tryptophan import. Additionally, Tat2I285V and Tat2I285T levels increased rapidly, and were efficiently localized to the cell surface after transferring the cells to low tryptophan medium (0.5 µg/mL). Our findings provide a clue to gain insights into the property of high-affinity transport mechanisms, and offer a unique approach to improve the functionality of broad types of amino acid permeases.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico , Cinética , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Triptofano/metabolismo
6.
Yeast ; 36(2): 85-97, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30350382

RESUMO

Reduction of gravity results in changes in gene expression and morphology in the budding yeast Saccharomyces cerevisiae. We studied the genes responsible for the morphological changes induced by simulated microgravity (SMG) using the yeast morphology data. We comprehensively captured the features of the morphological changes in yeast cells cultured in SMG with CalMorph, a high-throughput image-processing system. Statistical analysis revealed that 95 of 501 morphological traits were significantly affected, which included changes in bud direction, the ratio of daughter to mother cell size, the random daughter cell shape, the large mother cell size, bright nuclei in the M phase, and the decrease in angle between two nuclei. We identified downregulated genes that impacted the morphological changes in conditions of SMG by focusing on each of the morphological features individually. Gene Ontology (GO)-enrichment analysis indicated that morphological changes under conditions of SMG were caused by cooperative downregulation of 103 genes annotated to six GO terms, which included cytoplasmic ribonucleoprotein granule, RNA elongation, mitotic cell cycle phase transition, nucleocytoplasmic transport, protein-DNA complex subunit organization, and RNA localization. P-body formation was also promoted under conditions of SMG. These results suggest that cooperative downregulation of multiple genes occurs in conditions of SMG.


Assuntos
Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico , Ausência de Peso , Biometria , Perfilação da Expressão Gênica , Ontologia Genética , Processamento de Imagem Assistida por Computador , Imagem Óptica , Saccharomyces cerevisiae/genética
7.
Biochim Biophys Acta Biomembr ; 1859(10): 2076-2085, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28754537

RESUMO

Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Linhagem Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Polimorfismo de Nucleotídeo Único/genética , Triptofano/metabolismo , Tirosina/metabolismo , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo
8.
Biochim Biophys Acta Mol Cell Res ; 1864(2): 393-398, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27916534

RESUMO

Blasticidin S (BlaS) interferes in the cell growth of both eukaryotes and prokaryotes. Its mode of action as a protein synthesis inhibitor has been investigated extensively. However, the mechanism of BlaS transport into the target cells is not understood well. Here, we show that Ptr2, a member of the proton-dependent oligopeptide transporter (POT) family, is responsible for the uptake of BlaS in yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Notably, some mutants of Ptr2 that are dysfunctional in dipeptide uptake were still competent to transport BlaS. Mouse-derived oligopeptide transporter PepT1 conferred BlaS sensitivity in the S. cerevisiae ptr2∆ mutant. Furthermore, bacterial POT family proteins also potentiated the BlaS sensitivity of E. coli. The role of the POT family oligopeptide transporters in the uptake of BlaS is conserved across species from bacteria to mammals.


Assuntos
Antibacterianos/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Animais , Camundongos , Nucleosídeos/metabolismo , Transportador 1 de Peptídeos , Saccharomyces cerevisiae/metabolismo , Simportadores/fisiologia
9.
Subcell Biochem ; 72: 371-81, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26174391

RESUMO

Biological processes associated with dynamic structural features of membranes are highly sensitive to changes in hydrostatic pressure and temperature. Marine organisms potentially experience a broad range of pressure and temperature fluctuations. Hence, they have specialized cell membranes to perform membrane protein functions under various environmental conditions. Although the effects of high pressure on artificial lipid bilayers have been investigated in detail, little is known about how high pressure affects the structure of natural cell membranes and how organisms cope with pressure alterations. This review focused on the recent advances in research on the effects of high pressure on microbial membranes, particularly on the use of time-resolved fluorescence anisotropy measurement to determine membrane dynamics in deep-sea piezophiles.


Assuntos
Bactérias/química , Pressão Hidrostática , Saccharomyces cerevisiae/química , Membrana Celular/química , Ácidos Graxos Insaturados/química
10.
FEMS Yeast Res ; 15(6)2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26187908

RESUMO

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between -0.2 and -0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.


Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular , Eletrodos/microbiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Moléculas de Adesão Celular/genética , Deleção de Genes , Vidro , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Compostos de Estanho , Óxido de Zinco
11.
FEMS Yeast Res ; 15(5): fov044, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26071436

RESUMO

In Saccharomyces cerevisiae, high-affinity tryptophan import is performed by subtle mechanisms involving tryptophan permease Tat2. We have shown that Tat2 requires 15 amino acid residues in the transmembrane domains (TMDs) for its import activity, whereas leucine permease Bap2 requires only seven corresponding residues for its leucine import. For this reason, the structure of Tat2 is elaborately designed to transport the hydrophobic and bulky tryptophan. Newly synthesized cell surface proteins first undergo endoplasmic reticulum (ER)-associated quality check before entering the secretory pathway. In this study, we used domain replacement with general amino acid permease Gap1 to show that Tat2 chimeric proteins were dysfunctional when TMD10 or TMD11 was replaced. These chimeras formed large 270-800-kDa protein complexes and were stably retained in the ER membrane without efficient degradation. In contrast, Tat2 chimeras of TMD9 or TMD12 retained some of their tryptophan import activity and underwent vacuolar degradation as observed with wild-type Tat2. Thus, ours results suggest that TMD10 and TMD11 are essential for the correct folding of Tat2, probably because of their interdomain interactions. Notably, overexpression of Tat2-Gap1 chimera of TMD10 activated the unfolded protein response (UPR) element-lacZ reporter, suggesting that ER retention of the protein aggregates induces the UPR.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Retículo Endoplasmático/metabolismo , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Appl Environ Microbiol ; 81(11): 3688-98, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25795678

RESUMO

In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation.


Assuntos
Aspergillus/fisiologia , Grão Comestível/microbiologia , Aromatizantes/metabolismo , Glucosilceramidas/metabolismo , Membranas/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico/efeitos dos fármacos , Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Grão Comestível/metabolismo , Etanol/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
13.
Extremophiles ; 18(5): 853-63, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25108363

RESUMO

Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding ß-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three ß-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.


Assuntos
Proteínas de Bactérias/genética , Celulases/genética , Flavobacteriaceae/genética , Gammaproteobacteria/genética , Sedimentos Geológicos/microbiologia , Metagenoma , Xilosidases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Celulases/metabolismo , Flavobacteriaceae/enzimologia , Gammaproteobacteria/enzimologia , Genes Bacterianos , Água do Mar/microbiologia , Xilosidases/metabolismo
14.
Eukaryot Cell ; 13(11): 1380-92, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25172766

RESUMO

The peptide transporter Ptr2 plays a central role in di- or tripeptide import in Saccharomyces cerevisiae. Although PTR2 transcription has been extensively analyzed in terms of upregulation by the Ubr1-Cup9 circuit, the structural and functional information for this transporter is limited. Here we identified 14 amino acid residues required for peptide import through Ptr2 based on the crystallographic information of Streptococcus thermophilus peptide transporter PepTst and based on the conservation of primary sequences among the proton-dependent oligopeptide transporters (POTs). Expression of Ptr2 carrying one of the 14 mutations of which the corresponding residues of PepTst are involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enable ptr2Δtrp1 cell growth in alanyl-tryptophan (Ala-Trp) medium. We observed that Ptr2 underwent rapid degradation after cycloheximide treatment (half-life, approximately 1 h), and this degradation depended on Rsp5 ubiquitin ligase. The ubiquitination of Ptr2 most likely occurs at the N-terminal lysines 16, 27, and 34. Simultaneous substitution of arginine for the three lysines fully prevented Ptr2 degradation. Ptr2 mutants of the presumed peptide-binding site (E92Q, R93K, K205R, W362L, and E480D) exhibited severe defects in peptide import and were subjected to Rsp5-dependent degradation when cells were moved to Ala-Trp medium, whereas, similar to what occurs in the wild-type Ptr2, mutant proteins of the intracellular gate were upregulated. These results suggest that Ptr2 undergoes quality control and the defects in peptide binding and the concomitant conformational change render Ptr2 subject to efficient ubiquitination and subsequent degradation.


Assuntos
Sítios de Ligação/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas de Transporte/genética , Cristalografia por Raios X , Cicloeximida/farmacologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Membrana Transportadoras/ultraestrutura , Inibidores da Síntese de Proteínas/farmacologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação
15.
J Lipid Res ; 55(7): 1343-56, 2014 07.
Artigo em Inglês | MEDLINE | ID: mdl-24875539

RESUMO

In the yeast Saccharomyces cerevisiae, structural diversities of complex sphingolipids [inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide] are often observed in the presence or absence of hydroxyl groups on the C-4 position of long-chain base (C4-OH) and the C-2 position of very long-chain fatty acids (C2-OH), but the biological significance of these groups remains unclear. Here, we evaluated cellular membrane fluidity in hydroxyl group-defective yeast mutants by fluorescence recovery after photobleaching. The lateral diffusion of enhanced green fluorescent protein-tagged hexose transporter 1 (Hxt1-EGFP) was influenced by the absence of C4-OH and/or C2-OH. Notably, the fluorescence recovery of Hxt1-EGFP was dramatically decreased in the sur2Δ mutant (absence of C4-OH) under the csg1Δcsh1Δ background, in which mannosylation of IPC is blocked leading to IPC accumulation, while the recovery in the scs7Δ mutant (absence of C2-OH) under the same background was modestly decreased. In addition, the amount of low affinity tryptophan transporter 1 (Tat1)-EGFP was markedly decreased in the sur2Δcsg1Δcsh1Δ mutant and accumulated in intracellular membranes in the scs7Δcsg1Δcsh1Δ mutant without altering its protein expression. These results suggest that C4-OH and C2-OH are most probably critical factors for maintaining membrane fluidity and proper turnover of membrane molecules in yeast containing complex sphingolipids with only one hydrophilic head group.


Assuntos
Membrana Celular/metabolismo , Ceramidas/metabolismo , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/genética , Ceramidas/genética , Proteínas de Membrana/genética , Oxigenases de Função Mista/genética , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
16.
Biochim Biophys Acta ; 1838(7): 1719-29, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24699373

RESUMO

Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Leucina/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Sítios de Ligação , Leucina/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
17.
PLoS One ; 8(8): e73212, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24014139

RESUMO

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the ß-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure.


Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Glicina/metabolismo , Proteínas Luminescentes/metabolismo , Mutação de Sentido Incorreto , Proteínas de Bactérias/genética , Escherichia coli/genética , Glicina/genética , Pressão Hidrostática , Proteínas Luminescentes/genética
18.
Biophys Chem ; 183: 3-8, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23790318

RESUMO

High hydrostatic pressure has a profound physiological impact on lipid membranes, primarily resulting in tighter packing and restriction of acyl-chain motion. To fulfill membrane protein functions in high-pressure environments, deep-sea organisms possess specialized cell membranes. Although the effects of high-pressure on model membranes have been investigated in great detail, high-pressure-induced structural changes in living cell membranes remain to be elucidated. Of the spectroscopic techniques available to date, fluorescence anisotropy measurement is a common useful method that provides information on dynamic membrane properties. This mini-review focuses on pressure-induced changes in natural cell membranes, analyzed by means of high-pressure time-resolved fluorescence anisotropy measurement (HP-TRFAM). Specifically, the role of eicosapentaenoic acid in deep-sea piezophiles is described in terms of the structural integrity of the membrane under high pressure.


Assuntos
Membrana Celular/fisiologia , Pressão Hidrostática , Proteínas de Membrana/fisiologia , Membrana Celular/efeitos dos fármacos , Polarização de Fluorescência , Lipídeos de Membrana/farmacologia , Membranas Artificiais
19.
Biochemistry ; 52(25): 4296-307, 2013 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-23768406

RESUMO

Tryptophan is hydrophobic, bulky, and the rarest amino acid found in nutrients. Accordingly, the import machinery can be specialized evolutionarily. Our previous study in Saccharomyces cerevisiae demonstrated that tryptophan import by the high-affinity tryptophan permease Tat2 is accompanied by a large volume increase during substrate import. Nevertheless, the mechanisms by which the permease mediates tryptophan recognition and permeation remain to be elucidated. Here we determined amino acid residues essential for Tat2-mediated tryptophan import. By means of random mutagenesis in combination with site-directed mutagenesis based on crystallographic studies of the Escherichia coli arginine/agmatine antiporter AdiC, we identified 15 amino acid residues in the Tat2 transmembrane domains (TMDs) 1, -3, -5, -8, and -10, which are responsible for tryptophan uptake. T98, Y167, and E286 were assumed to form the central cavity in Tat2. G97/T98 and E286 were located within the putative α-helix break in TMD1 and TMD6, respectively, which are highly conserved among yeast amino acid permeases and bacterial solute transporters. Given the conformational change in AdiC upon substrate binding, G97/T98 and E286 of Tat2 were assumed to mediate a structural shift from an outward-open to a tryptophan-bound-occluded structure upon tryptophan binding, and T320, V322, and F324 became stabilized in TMD7. Such dynamic structural changes may account for the large volume increase associated with tryptophan import occurring concomitantly with a movement of water molecules from the tryptophan binding site. We also propose the working hypothesis that E286 mediates the proton influx that is coupled to tryptophan import.


Assuntos
Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Sistemas de Transporte de Aminoácidos/genética , Cristalografia por Raios X , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Triptofano/química
20.
Eukaryot Cell ; 12(7): 990-7, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23666621

RESUMO

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1(K29R-K31R)-GFP remained. The HPG1-1 (Rsp5(P514T)) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Pressão Hidrostática , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Pressão Atmosférica , Espaço Intracelular/metabolismo , Lisina/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Triptofano/metabolismo , Ubiquitinação
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