RESUMO
The C-terminal tail of yeast plasma membrane (PM) H(+)-ATPase extends approximately 38 amino acids beyond the final membrane-spanning segment (TM10) of the protein and is known to be required for successful trafficking, stability, and regulation of enzyme activity. To carry out a detailed functional survey of the entire length of the tail, we generated 15 stepwise truncation mutants. Eleven of them, lacking up to 30 amino acids from the extreme terminus, were able to support cell growth, even though there were detectable changes in plasma membrane expression, protein stability, and ATPase activity. Three functionally distinct regions of the C terminus could be defined. (i) Truncations upstream of Lys(889), removing more than 30 amino acid residues, yielded no viable mutants, and conditional expression of such constructs supported the conclusion that the stretch from Ala(881) (at the end of TM10) to Gly(888) is required for stable folding and PM targeting. (ii) The stretch between Lys(889) and Lys(916), a region known to be subject to kinase-mediated posttranslational modification, was shown here to be ubiquitinated in carbon-starved cells as part of cellular quality control and to be essential for normal ATPase folding and stability, as well as for autoinhibition of ATPase activity during glucose starvation. (iii) Finally, removal of even one or two residues (Glu(917) and Thr(918)) from the extreme C terminus led to visibly reduced expression of the ATPase at the plasma membrane. Thus, the C terminus is much more than a simple appendage and profoundly influences the structure, biogenesis, and function of the yeast H(+)-ATPase.
Assuntos
Dobramento de Proteína , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Ubiquitinação , Sequência de Aminoácidos , Membrana Celular/metabolismo , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Fungi, plants, and bacteria accumulate potassium via two distinct molecular machines not directly coupled to ATP hydrolysis. The first, designated TRK, HKT, or KTR, has eight transmembrane helices and is folded like known potassium channels, while the second, designated HAK, KT, or KUP, has 12 transmembrane helices and resembles MFS class proteins. One of each type functions in the model organism Neurospora crassa, where both are readily accessible for biochemical, genetic, and electrophysiological characterization. We have now determined the operating balance between Trk1p and Hak1p under several important conditions, including potassium limitation and carbon starvation. Growth measurements, epitope tagging, and quantitative Western blotting have shown the gene HAK1 to be much more highly regulated than is TRK1. This conclusion follows from three experimental results: (i) Trk1p is expressed constitutively but at low levels, and it is barely sensitive to extracellular [K(+)] and/or the coexpression of HAK1; (ii) Hak1p is abundant but is markedly depressed by elevated extracellular concentrations of K(+) and by coexpression of TRK1; and (iii) Carbon starvation slowly enhances Hak1p expression and depresses Trk1p expression, yielding steady-state Hak1p:Trk1p ratios of â¼500:1, viz., 10- to 50-fold larger than that in K(+)- and carbon-replete cells. Additionally, it appears that both potassium transporters can adjust kinetically to sustained low-K(+) stress by means of progressively increasing transporter affinity for extracellular K(+). The underlying observations are (iv) that K(+) influx via Trk1p remains nearly constant at â¼9 mM/h when extracellular K(+) is progressively depleted below 0.05 mM and (v) that K(+) influx via Hak1p remains at â¼3 mM/h when extracellular K(+) is depleted below 0.1 mM.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Neurospora crassa/metabolismo , Potássio/metabolismo , Western Blotting , Metabolismo dos Carboidratos , Proteínas de Transporte de Cátions/genética , Membrana Celular/metabolismo , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Neurospora crassa/crescimento & desenvolvimento , FenótipoRESUMO
Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P(2)-type ATPases, M8 has until now received the least attention. The present study has used alanine-scanning mutagenesis to map structure-function relationships throughout M8 of the yeast plasma-membrane H(+)-ATPase. Mutant forms of the ATPase were expressed in secretory vesicles and at the plasma membrane for measurements of ATP hydrolysis and ATP-dependent H(+) pumping. In secretory vesicles, Ala substitutions at a cluster of four positions near the extracytoplasmic end of M8 led to partial uncoupling of H(+) transport from ATP hydrolysis, while substitution of Ser-800 (close to the middle of M8) by Ala increased the apparent stoichiometry of H(+) transport. A similar increase has previously been reported following the substitution of Glu-803 by Gln (Petrov, V. et al., J. Biol. Chem. 275:15709-15718, 2000) at a position known to contribute directly to Ca(2+) binding in the Ca(2+)-ATPase of sarcoplasmic reticulum (Toyoshima, C., et al., Nature 405: 647-655, 2000). Four other mutations in M8 interfered with H(+)-ATPase folding and trafficking to the plasma membrane; based on homology modeling, they occupy positions that appear important for the proper bundling of M8 with M5, M6, M7, and M10. Taken together, these results point to a key role for M8 in the biogenesis, stability, and physiological functioning of the H(+)-ATPase.
Assuntos
Membrana Celular/enzimologia , ATPases Translocadoras de Prótons/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , ATPases Translocadoras de Prótons/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Relação Estrutura-AtividadeRESUMO
Given that many antifungal medications are susceptible to evolved resistance, there is a need for novel drugs with unique mechanisms of action. Inhibiting the essential proton pump Pma1p, a P-type ATPase, is a potentially effective therapeutic approach that is orthogonal to existing treatments. We identify NSC11668 and hitachimycin as structurally distinct antifungals that inhibit yeast ScPma1p. These compounds provide new opportunities for drug discovery aimed at this important target.
RESUMO
The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.
Assuntos
Antimaláricos/metabolismo , Indóis/metabolismo , ATPases do Tipo-P/metabolismo , Compostos de Espiro/metabolismo , Sequência de Aminoácidos , Antimaláricos/química , Antimaláricos/farmacologia , Sítios de Ligação , Sistemas CRISPR-Cas/genética , Citosol/química , Citosol/efeitos dos fármacos , Farmacorresistência Fúngica , Indóis/química , Indóis/farmacologia , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , ATPases do Tipo-P/antagonistas & inibidores , ATPases do Tipo-P/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Sequenciamento Completo do GenomaRESUMO
In the yeast plasma-membrane H(+)-ATPase and other P-type ATPases, conformational changes are transmitted between cytoplasmic and membrane-embedded domains via a stalk region composed of cytoplasmic extensions of membrane segments 2, 3, 4, and 5. The present study has used a fluorescent maleimide (Alexa-488) to probe Cys residues introduced into stalk segments 4 and 5 of the yeast enzyme. In the case of S5, Cys substitutions along one face led to a constitutive, 5- to 10-fold activation of the ATPase in the absence of glucose. Based on homology with SERCA Ca(2+)-ATPase, this face is likely to be buried in the interior of the protein, close to the P domain. Three Cys residues on the opposite face of S5 (A668C, S672C, and D676C) were accessible to Alexa-488 under all conditions tested. In addition, three other Cys residues at or near the boundary between the two faces reacted with Alexa-488 only (V665C, L678C) or preferentially (Y689C) in plasma membranes from glucose-metabolizing cells; this result provides the first direct evidence for a change in conformation of S5 during glucose activation. For stalk segment 4, site-directed mutagenesis gave no sign of a role in glucose-dependent regulation. Rather, substitutions at 13 consecutive positions along S4 caused kinetic changes consistent with a shift in equilibrium from E2 to E1. Four Cys residues along this stretch of S4 (Q357C, K362C, S364C, and S368C) reacted with Alexa-488, indicating that they are exposed to the aqueous medium as predicted in the SERCA-based structural model.
Assuntos
Corantes Fluorescentes/farmacologia , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Membrana Celular/enzimologia , Histidina , Cinética , Maleimidas/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H(+)-ATPase, which is responsible for H(+)-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896-918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal (14)N/(15)N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H(+)-ATPase.
Assuntos
Glucose/farmacologia , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Edulcorantes/farmacologia , Cromatografia Líquida de Alta Pressão , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Glucose/metabolismo , Peptídeos/metabolismo , Fosforilação , Estrutura Terciária de Proteína/fisiologia , Espectrometria de Massas por Ionização por Electrospray , Edulcorantes/metabolismoRESUMO
Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.
Assuntos
Membrana Celular/enzimologia , Fragmentos de Peptídeos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Deleção de Sequência , Transporte Biológico/genética , Membrana Celular/genética , Estabilidade Enzimática/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Dobramento de Proteína , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/química , Vacúolos/genética , Vacúolos/metabolismoRESUMO
Yeast Pma1 H(+)-ATPase, which belongs to the P-type family of cation-transporting ATPases, is activated up to 10-fold by growth on glucose, and indirect evidence has linked the activation to Ser/Thr phosphorylation within the C-terminal tail. We have now used limited trypsinolysis to map glucose-induced conformational changes throughout the 100 kDa ATPase. In the wild-type enzyme, trypsin cleaves first at Lys-28 and Arg-73 in the extended N-terminal segment (sites T1 and T2); subsequent cleavages occur at Arg-271 between the A domain and M3 (site T3) and at Lys-749 or Lys-754 in the M6-M7 cytoplasmic loop (site T4). Activation by glucose leads to a striking increase in trypsin sensitivity. At the C-terminal end of the protein, the Arg- and Lys-rich tail is shielded from trypsin in membranes from glucose-starved cells (GS) but becomes accessible in membranes from glucose-metabolizing cells (GM). In the presence of orthovanadate, Lys-174 at the boundary between M2 and the A domain also becomes open to cleavage in GM but not GS samples (site T5). Significantly, this global conformational change can be suppressed by mutations at Thr-912, a consensus phosphorylation site near the C-terminus. Substitution by Ala at position 912 leads to a GS-like (trypsin-resistant) state, while substitution by Asp leads to a GM-like (trypsin-sensitive) state. Thus, the present results help to dissect the intramolecular movements that result in glucose activation.
Assuntos
Glucose/farmacologia , ATPases Translocadoras de Prótons/química , Saccharomyces cerevisiae/enzimologia , Treonina/metabolismo , Membrana Celular/enzimologia , Ativação Enzimática , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Conformação Proteica , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Serina/genética , Treonina/química , Treonina/genética , Tripsina/químicaRESUMO
Glucose is well known to cause a rapid, reversible activation of the yeast plasma membrane H(+)-ATPase, very likely mediated by phosphorylation of two or more Ser/Thr residues near the C terminus. Recent mutagenesis studies have shown that glucose-dependent activation can be mimicked constitutively by amino acid substitutions in stalk segment 5 (S5), an alpha-helical stretch connecting the catalytic part of the ATPase with transmembrane segment 5 (Miranda, M., Allen, K. E., Pardo, J. P., and Slayman, C. W. (2001) J. Biol. Chem. 276, 22485-22490). In the present work, the fluorescent maleimide Alexa-488 has served as a probe for glucose-dependent changes in the conformation of S5. Experiments were carried out in a "3C" version of the ATPase, from which six of nine native cysteines had been removed by site-directed mutagenesis to eliminate background labeling by Alexa-488. In this construct, three of twelve cysteines introduced at various positions along S5 (A668C, S672C, and D676C) reacted with the Alexa dye in a glucose-independent manner, as shown by fluorescent labeling of the 100 kDa Pma1 polypeptide and by isolation and identification of the corresponding tryptic peptides. Especially significant was the fact that three additional cysteines reacted with Alexa-488 more rapidly (Y689C) or only (V665C and L678C) in plasma membranes from glucose-metabolizing cells. The results support a model in which the S5 alpha-helix undergoes a significant change in conformation to expose positions 665, 678, and 689 during glucose-dependent activation of the ATPase.
Assuntos
Corantes Fluorescentes/química , Glucose/metabolismo , Maleimidas/química , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Modelos Moleculares , Conformação Proteica , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The yeast plasma-membrane H(+)-ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway and has recently emerged as an excellent system for identifying quality control mechanisms along the pathway. In the present study, we have tracked the biogenesis of Pma1-G381A, a misfolded mutant form of the H(+)-ATPase. Although this mutant ATPase is arrested transiently in the peripheral endoplasmic reticulum, it does not become a substrate for endoplasmic reticulum-associated degradation nor does it appear to stimulate an unfolded protein response. Instead, Pma1-G381A accumulates in Kar2p-containing vesicular-tubular clusters that resemble those previously described in mammalian cells. Like their mammalian counterparts, the yeast vesicular-tubular clusters may correspond to specific exit ports from the endoplasmic reticulum, since Pma1-G381A eventually escapes from them (still in a misfolded, trypsin-sensitive form) to reach the plasma membrane. By comparison with wild-type ATPase, Pma1-G381A spends a short half-life at the plasma membrane before being removed and sent to the vacuole for degradation in a process that requires both End4p and Pep4p. Finally, in a separate set of experiments, Pma1-G381A was found to impose its phenotype on co-expressed wild-type ATPase, transiently retarding the wild-type protein in the ER and later stimulating its degradation in the vacuole. Both effects serve to lower the steady-state amount of wild-type ATPase in the plasma membrane and, thus, can explain the co-dominant genetic behavior of the G381A mutation. Taken together, the results of this study establish Pma1-G381A as a useful new probe for the yeast secretory system.