Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb+ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the Vmax of the low-affinity system has decreased from about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.     

Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb⁺ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the low-affinity system has decreased for about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.     

Positive feedback regulates switching of phosphate transporters in S. cerevisiae

The regulation of transporters by nutrient-responsive signaling pathways allows cells to tailor nutrient uptake to environmental conditions. We investigated the role of feedback generated by transporter regulation in the budding yeast phosphate-responsive signal transduction (PHO) pathway. Cells starved for phosphate activate feedback loops that regulate high- and low-affinity phosphate transport. We determined that positive feedback is generated by PHO pathway-dependent upregulation of Spl2, a negative regulator of low-affinity phosphate uptake. The interplay of positive and negative feedback loops leads to bistability in phosphate transporter usage--individual cells express predominantly either low- or high-affinity transporters, both of which can yield similar phosphate uptake capacity. Cells lacking the high-affinity transporter, and associated negative feedback, exhibit phenotypes that arise from hysteresis due to unopposed positive feedback. In wild-type cells, population heterogeneity generated by feedback loops may provide a strategy for anticipating changes in environmental phosphate levels.     

Adaptive changes in phosphate uptake by the fungus Neurospora crassa in response to phosphate supply.

The phosphate uptake rate of Neurospora crassa germlings growing exponentially in media containing phosphate at concentrations between 10 mM and 50 micronM was virtually constant. The uptake characteristics of these germlings were studied in detail assuming the simultaneous operation of two uptake systems, one of low affinity and one of high affinity. The Km of the low-affinity system was constant after growth at phosphate concentrations greater than 1 mM but became progressively lower as the concentration was reduced below 1 mM. In contrast, the Km of the high-affinity system was independent of the phosphate concentration of the growth medium. The Vmax of each system was highest after growth at low phosphate concentrations. As the phosphate concentration was increased to a maximum of 100 mM, the Vmax of the low-affinity system fell gradually, whereas that of the high-affinity system at first fell rapidly but then reached a constant minimum value at concentrations of 2.5 mM and higher. The differences in the kinetic parameters fully account for the constancy of uptake rate shown by the germlings.     

Effect of pH on the kinetics of Na+-dependent phosphate transport in rat renal brush-border membranes

The kinetics of Na+-dependent phosphate uptake in rat renal brush-border membrane vesicles were studied under zero-trans conditions at 37 degrees C and the effect of pH on the kinetic parameters was determined. When the pH was lowered it turned out to be increasingly difficult to estimate initial rates of phosphate uptake due to an increase in aspecific binding of phosphate to the brush border membrane. When EDTA or beta-glycerophosphate was added to the uptake medium this aspecific binding was markedly reduced. At pH 6.8, initial rates of phosphate uptake were measured between 0.01 and 3.0 mM phosphate in the presence of 100 mM Na+. Kinetic analysis resulted in a non-linear Eadie-Hofstee plot, compatible with two modes of transport: one major low-affinity system (Km approximately equal to 1.3 mM), high-capacity system (Vmax approximately equal to 1.1 nmol/s per mg protein) and one minor high-affinity (Km approximately equal to 0.03 mM), low-capacity system (Vmax approximately equal to 0.04 nmol/s per mg protein). Na+-dependent phosphate uptake studied far from initial rate conditions i.e. at 15 s, frequently observed in the literature, led to a dramatic decrease in the Vmax of the low-affinity system. When both the extra- and intravesicular pH were increased from 6.2 to 8.5, the Km value of the low-affinity system increased, but when divalent phosphate is considered to be the sole substrate for the low-affinity system then the Km value is no longer pH dependent. In contrast, the Km value of the high-affinity system was not influenced by pH but the Vmax decreased dramatically when the pH is lowered from 8.5 to 6.2. These results suggest that the low-affinity, high-capacity system transports divalent divalent phosphate only while the high-affinity, low-capacity system may transport univalent as well as divalent phosphate. Raising medium sodium concentration from 100 to 250 mM increased Na+-dependent phosphate uptake significantly but the pH dependence of the phosphate transport was not influenced. This observation makes it rather unlikely that pH changes only affect the Na+ site of the Na+-dependent phosphate transport system.     

Proton-linked transport systems as sensors of changes in the membrane surface potential

The kinetic properties of proton linked transport systems and their relation to the membrane surface potential were studied in yeast cells. (1) The negative surface potential of cells rich in anionic phospholipids was found to be 2-times higher than that of control cells; in agreement with their 2-fold increase in the anionic/zwitterionic phospholipid ratio (A/Z). (2) At low external concentration of substrates (high-affinity systems), higher uptake activities were observed for the anions, glutamate, aspartate and phosphate; the zwitterion glycine and the cations lysine and arginine, in both phosphatidylserine and phosphatidylinositol rich cells when compared to control cells. (3) On the other hand, at high external concentration of substrates (low-affinity systems), lower uptake activities were observed for glutamate, aspartate, phosphate and glycine in the cells rich in anionic phospholipids. (4) A decrease in Km without significant alteration in Vmax was found in the high-affinity transport systems that can be explained by the increase in proton concentration at the interface caused by the enhancement in negative surface charge of the cells rich in anionic phospholipids. (5) The mechanisms of the high-affinity proton linked transport systems are compatible with a model which is necessarily ordered, protons before anions. The low-affinity transport systems, on the other hand, follow a random order of binding. The transport systems studied behave as sensors of the changes in surface potential. The reduction of the surface potential reversed the transport alterations with the following sequence: monovalent cations less than divalent cations less than cationic local anesthetics.     

Kinetic characterization of the two phosphate uptake systems in the fungus Neurospora crassa.

The kinetics of phosphate uptake by exponentially growing Neurospora crassa were studied to determine the nature of the differences in uptake activity associated with growth at different external phosphate concentrations. Conidia, grown in liquid medium containing either 10 mM or 50 micronM phosphate, were harvested, and their phosphate uptake ability was measured. Initial experiments, where uptake was examined over a narrow concentration range near that of the growth medium, indicated the presence of a low-affintiy (high Km) system in germlings from 50 micronM phosphate. Uptake by each system was energy dependent and sensitive to inhibitors of membrane function. No efflux of phosphate or phosphorus-containing compounds could be detected. When examined over a wide concentration range, uptake was consistent with the simultaneous operation of low- and high-affinity systems in both types of germlings. The Vmax estimates for the two systems were higher in germlings from 50 micronM phosphate than for the corresponding systems in germlings from 10 mM phosphate. The Km of the high-affinity system was the same in both types of germlings, whereas the Km of the low-affinity system in germlings from 10 mM phosphate was about three three times that of the system in germlings from 50 micronM phosphate.     

Characterization of two phosphate transport systems in Acinetobacter johnsonii 210A.

The transport of P(i) was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P(i) is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P(i) limitation, contains two uptake systems with Kt values of 0.7 +/- 0.2 microM and 9 +/- 1 microM. P(i) uptake via the high-affinity component is drastically reduced by N,N'-dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, and by osmotic shock. Together with the presence of P(i)-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess P(i) to P(i)-free medium results in a 6- to 10-fold stimulation of the P(i) uptake rate. The constitutive low-affinity uptake system for P(i) is inhibited by uncouplers and can mediate counterflow of P(i), indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P(i) and the ability to decrease the free internal P(i) pool by forming polyP enable A. johnsonii 210A to reduce the P(i) concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and P(i) is released via the low-affinity secondary transport system.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.     

Modeling nitrogen uptake in oilseed rape cv Capitol during a growth cycle using influx kinetics of root nitrate transport systems and field experimental data

The use of kinetic equations of NO3- transport systems in oilseed rape (Brassica napus), determined by 15NO3- labeling under controlled conditions, combined with experimental field data from the INRA-Chalons rape database were used to model NO3- uptake during the plant growth cycle. The quantitative effects of different factors such as day/night cycle, ontogenetic stages, root temperature, photosynthetically active radiation, and soil nitrate availability on different components of the constitutive high-affinity transport systems, constitutive low-affinity transport systems, inducible low-affinity transport systems, and inducible high-affinity transport systems of nitrate were then determined to improve the model's predictions. Simulated uptake correlated well with measured values of nitrogen (N) uptake under field conditions for all N fertilization rates tested. Model outputs showed that the high-affinity transport system accounted for about 89% of total NO3- uptake (18% and 71% for constitutive high-affinity transport systems and inducible high-affinity transport systems, respectively) when no fertilizer was applied. The low-affinity transport system accounted for a minor proportion of total N uptake, and its activity was restricted to the early phase of the growth cycle. However, N fertilization in spring increased the duration of its contribution to total N uptake. Overall, data show that this mechanistic and environmentally regulated approach is a powerful means to simulate total N uptake in the field with the advantage of taking both physiologically regulated processes at the overall plant level and specific nitrate transport system characteristics into account.     

A novel alkali-tolerant Yarrowia lipolytica strain for dissecting Na+-coupled phosphate transport systems in yeasts

The newly isolated osmo-, salt- and alkali-tolerant Yarrowia lipolytica yeast strain is remarkable by its capacity to grow at alkaline pH values (pH 9.7), which makes it an excellent model system for studying Na(+)-coupled phosphate transport systems in yeast cells grown at alkaline conditions. In cells Y. lipolytica grown at pH 9.7, phosphate uptake was mediated by several kinetically discrete Na(+)-dependent systems that are specifically activated by Na(+) ions. One of these, a low-affinity transporter, operated at high-phosphate concentrations. The other two, derepressible, high-affinity, high-capacity systems, functioned during phosphate starvation. Both H(+)- and Na(+)-coupled high-affinity phosphate transport systems of Y. lipolytica cells were under the dual control of the prevailing extracellular phosphate concentrations and pH values. The contribution of the Na(+)/P(i)-cotransport systems into the total cellular phosphate uptake activity was progressively increased with increasing pH, reaching its maximum at pH > or = 9.     

Energetic consequences of two mutations in Escherichia coli K+ uptake systems for growth under potassium-limited conditions in the chemostat

The energetics of growth of two Escherichia coli strains (TK 2240 and TK 2242) differing in Km of the high-affinity potassium uptake system and lacking the low-affinity system were studied in the chemostat under potassium-limited conditions. The results were compared with the results obtained previously (Mulder, M.M., Teixeira de Mattos, M.J., Postma, P.W. and Van Dam, K. (1986) Biochim. Biophys. Acta 851, 223-228) with the wild-type FRAG-1, having two potassium uptake systems, and FRAG-5, a mutant which lacks the high-affinity potassium uptake system. We postulated that the high-affinity potassium uptake system was able to generate such a steep gradient across the membrane that the low-affinity system would act in reverse, thus creating a futile cycle of potassium ions at the cost of energy. As a result, FRAG-1 would show a higher ATP turnover at all growth rates tested than the mutant FRAG-5, in which strain the proposed futile cycle is interrupted because of the lack of the high-affinity system. It is shown here that the results obtained with TK 2240 and TK 2242 are in line with our hypothesis of futile potassium cycling. Under our experimental conditions, the yield on potassium was not dependent on the kinetic parameters of the uptake systems. The (thermodynamic) energy demand of the uptake systems determined the carbon substrate conversion required to achieve this yield.     

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis.     

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.     

Uptake of [N?Me?3H]-choline by synaptosomes from the central nervous system of Locusta migratoria

The accumulation of ³H-choline by isolated synaptosomes from the central nervous system of locust was studied at concentrations varying from 0.05 to 40μM. Kinetic analysis of the saturable process revealed a high-affinity and a low-affinity system. The high-affinity uptake was competitively inhibited by hemicholinium-3 and was absolutely dependent on external sodium. Elevated potassium concentrations inhibited choline uptake. The choline uptake by insect synaptosomes was found to be remarkably resistant to a variety of metabolic inhibitors. The reduced choline uptake under depolarizing conditions (high potassium concentration or veratridine) in the absence of calcium implies that electrochemical gradients are important for high-affinity choline uptake. Depolarization of preloaded synaptosomes under appropriate conditions resulted in a significant release of newly accumulated choline radioactivity.     

Phosphate-uptake systems in Yarrowia lipolytica cells grown under alkaline conditions

In this study we used a newly isolated Yarrowia lipolytica 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 phosphate transport systems in cells grown under alkaline conditions. Phosphate uptake by Y. lipolytica yeast cells grown at pH 9.5-10 was shown to be mediated by several kinetically discrete Na+-dependent systems. One of these, a low-affinity transporter, operates at high Pi concentrations and is, to our knowledge, here kinetically characterized for the first time. The other two high-affinity systems are derepressible, come into play under conditions of Pi-starvation, and appear to be controlled by the availability of extracellular Pi. They represent the first examples of high-capacity, Na+-driven Pi transport systems in an organism belonging to neither the animal nor the bacterial kingdoms.     

A new alkalitolerant <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeast strain is a promising model for dissecting properties and regulation of Na<Superscript>+</Superscript>-dependent phosphate transport systems

A newly isolated osmo-, salt-, and alkalitolerant Yarrowia lipolytica yeast strain is distinguished from other yeast species by its capacity to grow vigorously at alkaline pH values (9.7), which makes it a promising model organism for studying Na⁺-dependent phosphate transport systems in yeasts. Phosphate uptake by Y. lipolytica cells grown at pH 9.7 was mediated by several kinetically discrete Na⁺-dependent systems specifically activated by Na⁺. One of these, a low-affinity transporter, operated at high concentrations of extracellular phosphate. The other two, high-affinity systems, maximally active in phosphate-starved cells, were repressed or derepressed depending on the prevailing extracellular phosphate concentration and pH value. The contribution of Na⁺/P_i-cotransport systems to the total cellular phosphate uptake progressively increased with increasing pH, reaching its maximum at pH 9.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1607–1615.Original Russian Text Copyright © 2004 by Zvyagilskaya, Persson.     

High-Affinity Transport of γ-Aminobutyric Acid, Glycine, Taurine, L-Aspartic Acid, and L-Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.