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1.
Based on the high sequence homology between the yeast ORF YBR296c (accession number P38361 in the SWISS-PROT database) and the PHO4 gene of Neurospora crassa, which codes for a Na+/Pi cotransporter with twelve putative transmembrane domains, the YBR296c ORF was considered to be a promising candidate gene for a plasma membrane-bound phosphate transporter in Saccharomyces cerevisiae. Therefore, this gene, here designated PHO89, was cloned and a set of deletion mutants was constructed. We then studied their Pi uptake activity under different conditions. We show here that a transport activity displayed by PHO89 strains under alkaline conditions and in the presence of Na+ is absent in pho89 null mutants. Moreover, when the pH was lowered to pH 4.5 or when Na+ was omitted, this activity decreased significantly, reaching values close to those exhibited by the Δpho89 mutant. Studies of the acid phosphatase activity of these strains, as well as promoter sequence analysis, suggest that expression of the PHO89 gene is under the control of the PHO regulatory system. Northern analysis shows that this gene is only transcribed under conditions of Pi limitation. This is, to our knowledge, the first demonstration that the PHO89 gene codes for the Na+/Pi cotransporter previously characterized by kinetic studies, and represents the only Na+-coupled secondary anion transport system so far identified in S. cerevisiae. Pho89p has been shown to have an apparent Km of 0.5 μM and a pH optimum of 9.5, and is highly specific for Na+; activation of transport is maximal at a Na+ concentration of 25 mM. 相似文献
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4.
D H Lee K Uchiyama S Shioya Y I Hwang 《Journal of industrial microbiology & biotechnology》1998,20(3-4):160-165
In order to maximize the glucoamylase production by recombinant Saccharomyces cerevisiae in batch culture, first a temperature-controlled expression system for a foreign gene in S. cerevisiae was constructed. A temperature-sensitive pho80 mutant of S. cerevisiae for the PHO regulatory system, YKU131, was used for this purpose. A DNA fragment bearing the promoter of the PHO84 gene, which encodes an inorganic phosphate (Pi) transporter of S. cerevisiae and is derepressed by Pi starvation, was used as promoter. The glucoamylase gene connected with the PHO84 promoter was ligated into a YEp13 vector, designated pKU122. When the temperature-sensitive pho80
ts mutant harboring the plasmid pKU122 is cultivated at a lower temperature, the expression of glucoamylase gene is repressed,
but at a higher temperature it is expressed. Next the effect of temperature on the specific growth rate, μ, and specific production
rate, ρ, was investigated. Maximum values of ρ and ρ at various temperatures were at 30°C and 34°C, respectively. The optimal
cultivation temperature strategy for maximum production of glucoamylase by this recombinant strain in batch culture was then
determined by the Maximum principle using the relationships of μ and ρ to the cultivation temperature. Finally, the optimal
strategy was experimentally realized by changing the cultivation temperature from Tμ (30°C) to Tρ (34°C) at the switching
time, ts.
Received 18 September 1997/ Accepted in revised form 07 January 1998 相似文献
5.
The Na+/HCO3
− cotransporter is the main system that mediates bicarbonate removal out of the proximal tubule cell into the blood. We have
previously partially purified this protein and showed that chemical modification of the α-amino groups by fluorescein isothiocyanate
(FITC) inhibited the activity of the Na+/HCO3
− cotransporter. The inhibition was prevented by the presence of Na and bicarbonate suggesting that this compound binds at
or near the substrate transport sites of the cotransporter. We examined the effect of agents that modify the sulfhydryl group
(dithiothreitol), carboxyl groups (n-n′dicyclohexyl carbodiimide) and tyrosine residues (p-nitrobenzene sulfonyl fluoride, n-acetyl imidazole and tetranitromethane) on the activity of the cotransporter to gain insight into the chemical residues which
may be important for transport function. The sulfhydryl residues modifier, carboxyl group modifier, and tyrosine modifier
significantly inhibited bicarbonate dependent 22Na uptake in basolateral membranes by 50–70% without altering the 22Na uptake in the presence of gluconate indicating that these agents directly affected the cotransporter without affecting
diffusive sodium uptake. The effect of the tyrosine modifier n-acetylimidazole was not prevented by the presence of Na and bicarbonate suggesting that the tyrosine residues are not at
the substrate binding sites. To determine the presence and role of glycosylation on the Na+/HCO3
− cotransporter protein, we examined the effects of different glycosidases (endoglycosidase F and H, N-glycosidase F, O-glycanase) on the cotransporter activity. All glycosidases caused a significant 50–80% inhibition of cotransporter
activity. These data demonstrate that N-glycosylation as well as O-glycosylation are important for the function of the Na+/HCO3
− cotransporter protein. Taken together, these results suggest that chemical modifiers of tyrosine, carboxyl and sulfhydryl
groups as well as glycosylation are important for expression of full functional activity of the cotransporter.
Received: 8 October 1996/Revised: 23 January 1997 相似文献
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Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na+-dependent inorganic phosphate (Pi) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na+-dependent 32Pi cotransport in cultured fetal rat cortical neurons, where >90% of saturable Pi uptake is Na+-dependent. Saturable, Na+-dependent 32Pi uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na+-dependent Pi transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent Km values for Pi and Na+ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca2+ markedly reduced (>60%) Na+-dependent Pi uptake, with a threshold for maximal Pi import of 1–2.5 mM CaCl2. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na+ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na+. Furthermore, a substantial fraction of the 32Pi cotransported with Na+ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na+-dependent Pi import rates. These data establish the presence of a Na+-dependent Pi cotransport system in neurons of the CNS, demonstrate the Ca2+-dependent nature of 32Pi uptake, and suggest that the neuronal Na+-dependent Pi cotransporter may import Pi required for the production of high-energy compounds vital to neuronal metabolism. 相似文献
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The proximal tubule Na+-HCO−
3 cotransporter is located in the basolateral plasma membrane and moves Na+, HCO−
3, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO−
3 anions are transported for each Na+ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane
potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the
cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized
rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized
with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through
the cotransporter was achieved by applying Na+ or HCO−
3 concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference
current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl− independent; its conductance ranged between 0.12 and 0.23 mS/cm2 and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence
of Na+ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a
cotransport ratio of 3HCO−
3:1Na+.
Received: 19 January 1996/Revised: 24 April 1996 相似文献
10.
The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na+ rather than H+. To elucidate the mechanism of Na+ transport, the C-terminally truncated NuoL subunit (NuoLN) which is related to Na+/H+ antiporters was expressed as a protein A fusion protein (ProtA–NuoLN) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as
confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoLN catalyzed the uptake of Na+ and K+ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that
ProtA–NuoLN translocated Na+ and K+ independently from other complex I subunits. Na+ transport by ProtA–NuoLN was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na+/H+ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex
is discussed.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
11.
A.A. Bernardo F.T. Kear J.A. Stim O.S. Ruiz J.A.L. Arruda 《The Journal of membrane biology》1996,154(2):155-162
We have previously partially purified the basolateral Na+/HCO−
3 cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement
of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na+/HCO−
3 cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity.
Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that
the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against
the Na+/HCO−
3 cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na+/HCO−
3 cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes.
The purified antibody fraction caused a concentration-dependent inhibition of the Na+/HCO−
3 cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of
the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence
of the substrates (NaHCO3 or Na2CO3) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared
with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies
recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in
the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and
small intestine but was not detected in red blood cell membranes. Localization of the Na+/HCO−
3 cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes
of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against
the 56 kDa basolateral protein inhibit the activity of the Na+/HCO−
3 cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact
at or near the substrate binding sites. The Na+/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues.
Received: 27 January 1996/Revised: 23 July 1996 相似文献
12.
In the present study, we documented the promising role of thyroid hormones status in animals in modulation of Na+–Pi transport activity in intestinal brush border membrane vesicles (BBMV) which was accompanied with alterations in BBM lipid
composition and fluidity. Augmentation of net Pi balance in hyperthyroid (Hyper-T) rats was fraternized with accretion of Pi transport across BBMV isolated from intestine of Hyper-T rats as compared to hypothyroid (Hypo-T) and euthyroid (Eu-T) rats
while Na+–Pi transport across BBMV was decreased in Hypo-T rats relative to Eu-T rats. Increment in Na+–Pi transport in intestinal BBMV isolated from Hyper-T rats was manifested as an increase in the maximal velocity (Vmax) of Na+–Pi transport system. Furthermore, BBMV lipid composition profile in intestinal BBM from Hyper-T was altered to that of Hypo-T
rats and Eu-T rats. The molar ratio of cholesterol/phospholipids was higher in intestinal BBM from Hypo-T rats. Fluorescence
anistropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the intestinal BBM of Hyper-T rats
and decreased in Hypo-T rats as compared to Eu-T rats which corroborated with the alteration in membrane fluidity in response
to thyroid hormone status of animals. Therefore, thyroid hormone mediated change in membrane fluidity might play an important
role in modulating Na+–Pi transport activity of intestinal BBM. (Mol Cell Biochem 278: 195–202, 2005) 相似文献
13.
Inhibition of the ATPase by inorganic phosphate, Pi, was examined in terms of product inhibition of the various activities catalyzed by an enzyme preparation from rat brain, and considered in terms of the specific transport processes of the membrane Na+,K+-pump that these activities reflect. The K+-dependent phosphatase activity of the enzyme was most sensitive to Pi, and inhibition was competitive toward the substrate, nitrophenyl phosphate, as would be expected if Pi were released from the same enzyme form that bound substrate. However, this enzymatic activity does not seem to represent a transport process, and thus a cyclical discharge of K+ may not be involved. The Na+-dependent exchange activity was unaffected by Pi, in accord with the absence of Pi release in the reaction sequence. For the corresponding Na+/Na+ exchange function of the pump, which reportedly does not involve ATP hydrolysis either, prior release of Pi obviously cannot be required for Na+ discharge. With the Na+-dependent ATPase activity, measured using micromolar concentrations of ATP, Pi inhibited, but far less than with the phosphatase activity, and inhibition was not competitive toward ATP. Moreover, inhibition decreased as the Na+ concentration was raised from 10 to 100 mM. This elevated concentration of Na+ also led to substrate inhibition. For this ATPase activity, and the corresponding transport process, uncoupled Na+ efflux, the findings suggest that Na+ discharge follows Pi release, in contrast to Na+/Na+ exchange. The ATPase activity, measured with millimolar concentrations of ATP and reflecting the coupled Na+,K+-transport function, was similarly sensitive to Pi, and again inhibition was not competitive toward ATP. However, in this case inhibition did not increase as the Na+ concentration was lowered. For this activity, and the associated transport process, the site of Na+ discharge in the overall reaction sequence remains unresolved. 相似文献
14.
L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral
membrane vesicles l-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective.
The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is
saturable with respect to external lactate with a K
m
of 39.2 ± 4.8 mm and a J
max of 8.9 ± 0.7 nmoles mg protein−1 sec−1. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains
a H+-lactate cotransporter, whereas in the apical membrane both H+-lactate and Na+-lactate cotransporters are present, even if they exhibit a low transport rate.
Received: 22 October 1996/Revised: 11 March 1997 相似文献
15.
M. García-Delgado 《生物化学与生物物理学报:生物膜》2007,1768(11):2841-2848
16.
High salinity is an environmental factor that inhibits plant growth and development, leading to large losses in crop yields.
We report here that mutations in SIZ1 or PHO2, which cause more accumulation of phosphate compared with the wild type, enhance tolerance to salt stress. The siz1 and pho2 mutations reduce the uptake and accumulation of Na+. These mutations are also able to suppress the Na+ hypersensitivity of the sos3-1 mutant, and genetic analyses suggest that SIZ1 and SOS3 or PHO2 and SOS3 have an additive effect on the response to salt stress. Furthermore, the siz1 mutation cannot suppress the Li+ hypersensitivity of the sos3-1 mutant. These results indicate that the phosphate-accumulating mutants siz1 and pho2 reduce the uptake and accumulation of Na+, leading to enhanced salt tolerance, and that, genetically, SIZ1 and PHO2 are likely independent of SOS3-dependent salt signaling. 相似文献
17.
The transport activity and substrate specificity of two chimeras consisting of S. cerevisiae Nha1p’s N-terminal regions (either first 125 or 184 AA) and the rest of the C. glabrata Cnh1p (up to the total protein length of 946 AA) were compared with those of the two native antiporters. Both chimeric transporters
were functional upon expression in S. cerevisiae cells, their presence improved the ability of cells to grow in the presence of high external concentration of K+, Na+ or Rb+ (as chlorides), but not in the presence of the smallest cation (Li+). Cation efflux confirmed the ability of chimeras to export cations and showed their significantly reduced transport capacity
compared to the wild-type proteins. Despite the very high level of primary sequence identity (87 %) between the S. cerevisiae and C. glabrata plasma-membrane Na+/H+ antiporters, various parts of these proteins are not exchangeable without affecting the antiporter’s transport capacity. 相似文献
18.
Zvyagilskaya R Parchomenko O Abramova N Allard P Panaretakis T Pattison-Granberg J Persson BL 《The Journal of membrane biology》2001,183(1):39-50
In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5–10.5), which makes it an excellent model system for
studying H+- and Na+-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline
pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation.
This was demonstrated for the first time for cells grown at pH 9.5–10.0. In cells grown at pH 4.5, inorganic phosphate (Pi) was accumulated by two kinetically discrete H+/Pi-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high Pi concentrations. The high-affinity system, subjected to regulation by both extracellular Pi availability and intracellular polyphosphate stores, is mobilized during Pi-starvation. In cells grown at pH 9.5–10, Pi uptake is mediated by several kinetically discrete Na+-dependent systems that are specifically activated by Na+ ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high Pi concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems,
are derepressible and functional during Pi-starvation and appear to be controlled by extracellular Pi. They represent the first examples of high-capacity, Na+-driven Pi transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H+- and Na+-coupled Pi transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H+-coupled Pi transport systems are predominant. The contribution of the Na+/Pi cotransport systems to the total cellular Pi uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher.
Received: 15 December 2000/Revised: 14 May 2001 相似文献
19.
I.C. Forster C.A. Wagner A.E. Busch F. Lang J. Biber N. Hernando H. Murer A. Werner 《The Journal of membrane biology》1997,160(1):9-25
The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties
of the type II Na+/P
i
cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state P
i
-induced currents had a voltage-independent apparent K
m
for P
i
of 0.03 mm and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mm Na+. The apparent K
m
for Na+ at 1 mm P
i
was strongly voltage dependent (increasing from 32 mm at −70 mV to 77 mm at −30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na+ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage
jumps elicited presteady-state relaxations in the presence of 96 mm Na+ which were suppressed at saturating P
i
(1 mm). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at
extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically
gave a midpoint voltage (V
0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly
with the maximum P
i
-suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated
as 35 sec−1. The voltage dependence of the relaxations was P
i
-independent, whereas changes in Na+ shifted V
0.5 to −60 mV at 25 mm Na+. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na+. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions
involving reorientation of the unloaded carrier and binding of Na+ contribute to transmembrane charge movement.
Received: 11 March 1997/Revised: 3 June 1997 相似文献
20.
The type IIa Na+/Pi, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na+ and one divalent Pi ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms
with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant
of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected
it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and Pi transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally
altered kinetics of Pi transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared
to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or
N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the Pi-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in
the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na+-dependent Pi transport. Since electrogenic NaPi-II transports 3 Na+/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might
result from the loss of one of the three Na+ binding sites in NaPi-IIa. 相似文献