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1.
J Biol Chem ; 298(12): 102672, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36334632

RESUMO

Yeast vacuoles are acidified by the v-type H+-ATPase (V-ATPase) that is comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme on the vacuole is stabilized in part through interactions between the VO a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). PI(3,5)P2 also affects vacuolar Ca2+ release through the channel Yvc1 and uptake through the Ca2+ pump Pmc1. Here, we asked if H+ and Ca2+ transport activities were connected through PI(3,5)P2. We found that overproduction of PI(3,5)P2 by the hyperactive fab1T2250A mutant augmented vacuole acidification, whereas the kinase-inactive fab1EEE mutant attenuated the formation of a H+ gradient. Separately, we tested the effects of excess Ca2+ on vacuole acidification. Adding micromolar Ca2+ blocked vacuole acidification, whereas chelating Ca2+ accelerated acidification. The effect of adding Ca2+ on acidification was eliminated when the Ca2+/H+ antiporter Vcx1 was absent, indicating that the vacuolar H+ gradient can collapse during Ca2+ stress through Vcx1 activity. This, however, was independent of PI(3,5)P2, suggesting that PI(3,5)P2 plays a role in submicromolar Ca2+ flux but not under Ca2+ shock. To see if the link between Ca2+ and H+ transport was bidirectional, we examined Ca2+ transport when vacuole acidification was inhibited. We found that Ca2+ transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca2+ transport and V-ATPase efficacy are connected but not necessarily through PI(3,5)P2.


Assuntos
Proteínas de Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fosfatidilinositóis , Vacúolos/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
2.
Anal Biochem ; 658: 114927, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36167157

RESUMO

Eukaryotic cells are compartmentalized into membrane-bound organelles, allowing each organelle to maintain the specialized conditions needed for their specific functions. One of the features that change between organelles is lumenal pH. In the endocytic and secretory pathways, lumenal pH is controlled by isoforms and concentration of the vacuolar-type H+-ATPase (V-ATPase). In the endolysosomal pathway, copies of complete V-ATPase complexes accumulate as membranes mature from early endosomes to late endosomes and lysosomes. Thus, each compartment becomes more acidic as maturation proceeds. Lysosome acidification is essential for the breakdown of macromolecules delivered from endosomes as well as cargo from different autophagic pathways, and dysregulation of this process is linked to various diseases. Thus, it is important to understand the regulation of the V-ATPase. Here we describe a high-throughput method for screening inhibitors/activators of V-ATPase activity using Acridine Orange (AO) as a fluorescent reporter for acidified yeast vacuolar lysosomes. Through this method, the acidification of purified vacuoles can be measured in real-time in half-volume 96-well plates or a larger 384-well format. This not only reduces the cost of expensive low abundance reagents, but it drastically reduces the time needed to measure individual conditions in large volume cuvettes.


Assuntos
Laranja de Acridina , ATPases Vacuolares Próton-Translocadoras , Vacúolos , Endossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Lisossomos/metabolismo , Concentração de Íons de Hidrogênio
3.
Proc Natl Acad Sci U S A ; 116(15): 7272-7277, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30910982

RESUMO

Proton-translocating vacuolar-type ATPases (V-ATPases) are necessary for numerous processes in eukaryotic cells, including receptor-mediated endocytosis, protein maturation, and lysosomal acidification. In mammals, V-ATPase subunit isoforms are differentially targeted to various intracellular compartments or tissues, but how these subunit isoforms influence enzyme activity is not clear. In the yeast Saccharomyces cerevisiae, isoform diversity is limited to two different versions of the proton-translocating subunit a: Vph1p, which is targeted to the vacuole, and Stv1p, which is targeted to the Golgi apparatus and endosomes. We show that purified V-ATPase complexes containing Vph1p have higher ATPase activity than complexes containing Stv1p and that the relative difference in activity depends on the presence of lipids. We also show that VO complexes containing Stv1p could be readily purified without attached V1 regions. We used this effect to determine structures of the membrane-embedded VO region with Stv1p at 3.1-Å resolution, which we compare with a structure of the VO region with Vph1p that we determine to 3.2-Å resolution. These maps reveal differences in the surface charge near the cytoplasmic proton half-channel. Both maps also show the presence of bound lipids, as well as regularly spaced densities that may correspond to ergosterol or bound detergent, around the c-ring.

4.
Curr Genet ; 63(6): 1093-1104, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28560585

RESUMO

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding ß-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of ß-1, 3-glucan synthesis in C. neoformans; the inhibition of ß-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized ß-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.


Assuntos
Cryptococcus neoformans/genética , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Regulação Fúngica da Expressão Gênica , Glucosiltransferases/genética , Lipopeptídeos/farmacologia , ATPases Vacuolares Próton-Translocadoras/genética , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/crescimento & desenvolvimento , Ergosterol/biossíntese , Ergosterol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Engenharia Genética , Glucosiltransferases/deficiência , Micafungina , Testes de Sensibilidade Microbiana , Mutação , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , ATPases Vacuolares Próton-Translocadoras/deficiência
5.
Autophagy Rep ; 1(1): 226-233, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37389034

RESUMO

A significant number of follicular lymphoma patients display recurrent mutations in subunits and regulators of the vacuolar-type H+-translocating ATPase (V-ATPase). Past studies focusing on the role of these mutations highlighted essential functions of macroautophagy/autophagy, amino-acid, and nutrient-sensing pathways in the pathogenesis of this disease. Here, we demonstrate novel results understanding the role of the follicular lymphoma-associated hotspot mutation VMA21p.93X, which corresponds to Vma21[Δ66-77] in S. cerevisiae cells. We find that V-ATPase assembly is affected by the Vma21[Δ66-77] mutation, shown by decreased vacuolar levels of V0 subunits as well as a Vph1 stability assay. In addition, we report that vacuolar levels of histidine, lysine and arginine are significantly reduced in Vma21[Δ66-77] mutant cells. These results deepen the current understanding on the mechanism of how autophagy is activated by these mutations in follicular lymphoma.

6.
J Gen Appl Microbiol ; 67(5): 195-206, 2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34219070

RESUMO

We clarified the roles of VPH1 in Cryptococcus neoformans serotype D by examining the detailed phenotypes of VPH1-deficient cells (Δvph1) in terms of their capability to grow in acidic and alkaline pH, at a high temperature, and under high osmotic conditions, in addition to the involvement of VPH1 in copper (Cu) homeostasis and the expression of some C. neoformans virulence factors. Δvph1 could grow well on minimal medium (YNB) but exhibited hypersensitivity to 20 µM Cu due to the failure to induce Cu-detoxifying metallothionein genes (CMT1 and CMT2). In contrast, Δvph1 exhibited defective growth on rich medium (YPD), and the induction of Cu transporter genes (CTR1 and CTR4) did not occur in this medium, implying that this strain was incapable of the uptake of Cu ions for growth. However, the addition of excess Cu promoted CTR gene expression and supported Δvph1 growth. These results suggested that the lack of the VPH1 gene disturbed Cu homeostasis in C. neoformans. Moreover, the loss of Vph1 function influenced the urease activity of C. neoformans.


Assuntos
Proteínas de Bactérias/metabolismo , Cobre/fisiologia , Cryptococcus neoformans/fisiologia , Cryptococcus neoformans/genética , Homeostase , Sorogrupo
7.
BMC Mol Cell Biol ; 21(1): 70, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028189

RESUMO

BACKGROUND: Microautophagy, which degrades cargos by direct lysosomal/vacuolar engulfment of cytoplasmic cargos, is promoted after nutrient starvation and the inactivation of target of rapamycin complex 1 (TORC1) protein kinase. In budding yeast, microautophagy has been commonly assessed using processing assays with green fluorescent protein (GFP)-tagged vacuolar membrane proteins, such as Vph1 and Pho8. The endosomal sorting complex required for transport (ESCRT) system is proposed to be required for microautophagy, because degradation of vacuolar membrane protein Vph1 was compromised in ESCRT-defective mutants. However, ESCRT is also critical for the vacuolar sorting of most vacuolar proteins, and hence reexamination of the involvement of ESCRT in microautophagic processes is required. RESULTS: Here, we show that the Vph1-GFP processing assay is unsuitable for estimating the involvement of ESCRT in microautophagy, because Vph1-GFP accumulated highly in the prevacuolar class E compartment in ESCRT mutants. In contrast, GFP-Pho8 and Sna4-GFP destined for vacuolar membranes via an alternative adaptor protein-3 (AP-3) pathway, were properly localized on vacuolar membranes in ESCRT-deficient cells. Nevertheless, microautophagic degradation of GFP-Pho8 and Sna4-GFP after TORC1 inactivation was hindered in ESCRT mutants, indicating that ESCRT is indeed required for microautophagy after nutrient starvation and TORC1 inactivation. CONCLUSIONS: These findings provide evidence for the direct role of ESCRT in microautophagy induction.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Microautofagia/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Fermento Seco/metabolismo , Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Vacúolos/metabolismo
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