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.     

Vanadate uptake in Neurospora crassa occurs via phosphate transport system II.

Vanadate, a potent inhibitor of plasma membrane ATPases, is taken up by Neurospora crassa only when cells are growing in alkaline medium and starving for phosphate. The appearance of a vanadate uptake system (Km = 8.2 microM; Vmax = 0.15 mmol/min per liter of cell water) occurs under the same conditions required for derepression of a high-affinity phosphate transport system. Phosphate is a competitive inhibitor of vanadate uptake, and vanadate is a competitive inhibitor of phosphate uptake. Furthermore, mutant strains which are either partially constitutive or non-derepressible for the high-affinity phosphate transport system are also partially constitutive or non-derepressible for vanadate uptake. These data indicate that vanadate enters the cell via phosphate transport system II.     

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.     

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.     

Expression of kinase-dependent glucose uptake in Saccharomyces cerevisiae

There are both low- and high-affinity mechanisms for uptake of glucose in Saccharomyces cerevisiae; high-affinity uptake somehow depends on the presence of hexose kinases (L. F. Bisson and D. G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983; L. F. Bisson and D. G. Fraenkel, J. Bacteriol. 155:995-1000, 1983). We report here on the effect of culture conditions on the level of high-affinity uptake. The high-affinity component was low during growth in high concentrations of glucose (100 mM), increased as glucose was exhausted from the medium, and decreased again during prolonged incubation in the stationary phase. The higher level of uptake was found in growth on low concentrations of glucose (0.5 mM) and in growth on normal concentrations of galactose, lactate plus glycerol, or ethanol. These results suggest that some component of high-affinity uptake is repressible by glucose. A shift from medium with 100 mM glucose to medium with 5 mM glucose resulted in up to a 10-fold increase in the level of high-affinity uptake within 90 min; the increase did not occur in the presence of cycloheximide or 2,4-dinitrophenol or in buffer alone with low glucose, suggesting that protein synthesis or energy metabolism (or both) was required. Reimposition of the high glucose concentration caused loss of high-affinity uptake, a process not prevented by cycloheximide. The use of hexokinase single-gene mutants showed that the derepression of high-affinity uptake was not clearly correlated with changes in levels of the kinases themselves. These results place the phenomenon of high- and low-affinity uptake in a physiological context, in that high-affinity uptake seems to be expressed best in conditions where it might be needed. Apparent similarities between glucose uptake in yeast and animal cells are noted.     

Vanadate-resistant mutants of Candida albicans show alterations in phosphate uptake

Phosphate uptake studies in different strains of the dimorphic pathogenic yeast Candida albicans were undertaken to show that this yeast actively transported phosphate with an apparent Km in the range of 90-170 microM. The uptake was pH dependent and derepressible under phosphate starvation. Vanadate-resistant (van) mutants of C. albicans showed a 20-70% reduction in the rate of phosphate uptake in high phosphate medium and was associated with an increased Km and reduced Vmax. The magnitude of derepression under phosphate starvation was different between van mutants. These results demonstrate that van mutants may have developed resistance by modifying the rate of entry of vanadate.     

Effects of physiological manipulation on the kinetics of mitochondrial phosphate transport in Saccharomyces cerevisiae

The kinetics of [³²P]phosphate uptake has been studied in different types of Saccharomyces cerevisiae mitochondria. Mitochondria were isolated from yeast grown aerobically on 2% lactate (Lac-mitochondria), 2% galactose (Gal-mitochondria), 5.4% glucose (Glu-mitochondria) or from yeast grown anaerobically on 2% galactose (Promitochondria). The effect of chloramphenicol was also studied by adding it to the growth medium of yeast grown aerobically on 2% galactose (chloramphenicol-mitochondria).[³²P]Phosphate uptake followed an oscillatory pattern in Lac, Gal-mitochondria and Promitochondria.Saturation kinetics were detected in fully differenciated mitochondria and in Promitochondria, but not in chloramphenicol-mitochondria.Glu-mitochondria did not translocate phosphate as shown both by lack of [³²P]phosphate uptake and lack of swelling in isoosmotic potassium solution.Repressed yeast cells were incubated in a resting cell medium and mitochondria were isolated at different times of incubation. The rate of respiration and the oligomycin-sensitive ATPase increased during the course of the incubation. After 2h, a mitochondrial mersalyl-sensitive swelling in an isoosmotic potassium phosphate solution was detected.As expected, no increase of the rate of respiration was observed when chloramphenicol was added in the derepression medium. But the oligomycin-sensitive ATPase decreased. Chloramphenicol did not affect the phosphate transport activity as measured by the swelling of mitochondria, but the [³²P]phosphate uptake did not follow saturation kinetics. A complete derepression of the inorganic phosphate-carrier activity was achieved by a 4 h incubation of the repressed cells in the presence of chloramphenicol, followed by a 6 h incubation in presence of cycloheximide.These data strongly suggest that the mitochondrial protein-synthesis system is required for the normal function of the inorganic phosphate-carrier.     

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

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 mitochondrial functions on synthesis of yeast cytochrome c

The effects of the mitochondrial protein synthesis inhibitor chloramphenicol and the mitochondrial F0 adenosine triphosphatase inhibitor oligomycin on the synthesis of nucleus-encoded cytochrome c protein were studied. Both inhibitors stimulated cytochrome c protein synthesis in the derepressed state (growth in media containing 2% raffinose) but had no effect on the synthesis of the cytochrome c protein in the repressed state (growth in media containing 5% glucose). Oligomycin uncoupled the synthesis of the apoprotein from its processing into the hemoprotein. Neither antibiotic had a significant effect on the rate of glucose repression of cytochrome protein synthesis. The kinetics of cytochrome c derepression and the effects of these two antibiotics on these kinetics were also studied. Cells were derepressed by transfer from glucose- to faffinose-containing media, and the rate of cytochrome c synthesis increased from the repressed to the derepressed level during the second hour of derepression. Chloramphenicol delayed this derepression, but after 5 h the rate of cytochrome c protein synthesis increased to twice the rate of synthesis in uninhibited cells. On the other hand, oligomycin inhibited derepression of cytochrome c. These results are discussed with respect to the effects of mitochondrial function in the derepressed and repressed states and during the processes of repression and derepression of cytochrome c.     

Differential translational efficiency of the mRNAs isolated from derepressed and glucose repressed Saccharomyces cerevisiae

Carbon catabolite derepression induced changes in the pool of yeast mRNAs translatable in a protein-synthesizing reticulocyte system. Competition experiments with globin mRNA showed that the mRNA population obtained from derepressed cells possessed a higher translational efficiency than mRNA from repressed cells. The mRNAs that could account for the high translational efficiency of the derepressed mRNA were not detected in cells growing in glucose-rich medium. Analysis of protein synthesis in the presence of 7-methylguanosine 5'-phosphate indicated that the initiation factors recognizing the 5'-terminal structure of capped messengers interacted with lower affinity with the repressed than with some specific derepressed mRNAs.     

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.     

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 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.     

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.     

Investigation of transport-associated phosphorylation of sugar in yeast mutants (snf3) lacking high-affinity glucose transport and in a mutant (fdp1) showing deficient regulation of initial sugar metabolism

Pool-labeling experiments with 2-deoxyglucose in derepressed cells of the yeastSaccharomyces cerevisiae confirmed the previously reported results pointing to the possible existence of transport-associated phosphorylation of sugar. In yeast mutants containing a disruption or an inactivating point mutation in thesnf3 gene, which codes for the high-affinity glucose carrier, no evidence for transport-associated phosphorylation of 2-deoxyglucose was observed. If transport-associated phosphorylation in yeast exists, it is apparently not mediated by the low-affinity glucose carrier. Mediation by the high-affinity carrier would fit with the known requirement of an active kinase for high-affinity sugar transport. A mixed type of uptake in cells having both carriers would explain many of the problems associated with the 2-deoxyglucose pool-labeling experiments. Since mutants that have only low-affinity glucose transport are not deficient in the glucose-induced RAS-mediated cAMP signal, transport-associated phosphorylation of glucose is not required for or involved in the induction of the signal. The yeastfdp mutant, which dies on media containing fermentable sugars because of overaccumulation of sugar phosphates, also did not show any evidence for the existence of transport-associated phosphorylation. The same was true for the double mutantfdp snf3. The latter also showed the typicalfdp phenotype, indicative that the lethality on media containing fermentable sugar is owing to aberrant regulation of low-affinity transport. The high protein kinase activity in thefdp mutant does not appear to be responsible for the absence of evidence for transport-associated phosphorylation, because another mutant with high protein kinase activity, thebcy mutant, displayed normal transport behavior.     

Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.

The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western blot analysis, respectively. As detected by indirect immunofluorescence and antibody capture assay, both wild-type and mutant NaPi-2 proteins are expressed in the yeast plasma membrane in comparable amounts. In the presence of 5 microM phosphate, Na+ promotes phosphate uptake into yeast cells expressing the wild-type NaPi-2 with a K(0.5) of 5.6 +/- 1.1 mM. The maximum uptake of phosphate (649 +/- 30 pmol/10 min) is approximately 8-fold higher than the uptake obtained with nontransformed cells (76.8 +/- 8 pmol/10 min). Yeast cells expressing the R215E mutant of NaPi-2 accumulate 213 +/- 9 pmol of phosphate/10 min under the same conditions. The K(0.5) for the stimulation of phosphate uptake by Na+ is 4.2 +/- 0.8 mM for the R215E mutant and thus not significantly different from the value obtained with cells expressing the wild-type cotransporter. The reduced level of accumulation of phosphate in yeast cells expressing the R215E mutant is probably due to a reduction of the first-order rate constant k for phosphate uptake: while cells expressing wild-type NaPi-2 accumulate phosphate with a k of 0.06 min(-1), the rate for phosphate uptake into cells expressing the R215E mutant (k) is 0.016 min(-1) and therefore about 4-fold lower. In comparison, the rate for phosphate uptake into nontransformed cells (k) is 0.0075 min(-1). Phosphate uptake into yeast cells that express the wild-type NaPi-2 in the presence of 150 mM NaCl is promoted by extracellular phosphate with a K(0.5) of 45 +/- 4 microM. A phosphate-dependent phosphate accumulation is also observed with cells expressing the R215E mutant, but the K(0.5) is twice as high (86 +/- 5 microM) as that obtained with the wild-type cotransporter. We conclude that the yeast expression system is a useful tool for the investigation of structure-function relationships of the renal sodium/phosphate cotransporter and that (215)R, although not involved in Na+ recognition, is a part of the structure involved in phosphate recognition and considerably influences the rate of phosphate uptake by the NaPi-2 cotransporter.     

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.