Bengt L. Persson

Functional analysis and cell-specific expression of a phosphate transporter from tomato.

Abstract. For a better understanding of the molecular and biochemical processes involved in orthophosphate (Pi) uptake at the root/soil interface, we cloned a Pi-transporter cDNA (LePT1) from a root air-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The corresponding protein belongs to the growing family of ion transporters with twelve putative transmembrane domains. It is highly homologous to recently isolated Pi transporters from higher plants, yeast and fungi. When expressed in a Pi-uptake-deficient yeast mutant, the L. esculentum phosphate transporter 1 (LePT1) protein exhibits an apparent Km of 31 μM. The transporter is still active at submicromolar Pi concentrations and mediates highest Pi uptake at pH 5. The activity of LePT1 is dependent on the electrochemical membrane potential mediated by the yeast P-type H+-ATPase. Transcript levels of LePT1 in tomato seedlings are detectable in all vegetative organs under Pi-sufficient conditions, with highest concentrations in root hairs. In␣situ hybridization studies demonstrate cell-specific expression of LePT1 in the tomato root. The LePT1 mRNA is detectable in peripheral cell layers such as rhizodermal and root cap cells. Under Pi-deprivation conditions, mRNA levels are also detectable in young stelar tissue. This work presents molecular and biochemical evidence for distinct root cells playing an important role in Pi acquisition at the root/soil interface.

Regulation of phosphate acquisition in Saccharomyces cerevisiae

Abstract Membrane transport systems active in cellular inorganic phosphate (Pi) acquisition play a key role in maintaining cellular Pi homeostasis, independent of whether the cell is a unicellular microorganism or is contained in the tissue of a higher eukaryotic organism. Since unicellular eukaryotes such as yeast interact directly with the nutritious environment, regulation of Pi transport is maintained solely by transduction of nutrient signals across the plasma membrane. The individual yeast cell thus recognizes nutrients that can act as both signals and sustenance. The present review provides an overview of Pi acquisition via the plasma membrane Pi transporters of Saccharomyces cerevisiae and the regulation of internal Pi stores under the prevailing Pi status.

Intracellular localization of an active green fluorescent protein-tagged Pho84 phosphate permease in Saccharomyces cerevisiae.

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the carboxy‐terminus of the Pho84 phosphate permease of Saccharomyces cerevisiae. Both components of the fusion protein displayed their native functions and revealed a cellular localization and degradation of the Pho84‐GFP chimera consistent with the behavior of the wild‐type Pho84 protein. The GFP‐tagged chimera allowed for a detection of conditions under which the Pho84 transporter is localized to its functional environment, i.e. the plasma membrane, and conditions linked to relocation of the protein to the vacuole for degradation. By use of the methodology described, GFP should be useful in studies of localization and degradation also of other membrane proteins in vivo.

Phosphate permeases of Saccharomyces cerevisiae

The PHO84 and PHO89 genes of Saccharomyces cerevisiae encode two high-affinity phosphate cotransporters of the plasma membrane. Hydropathy analysis suggests a secondary structure arrangements of the proteins in 12 transmembrane domains. The derepressible Pho84 and Pho89 transporters appear to have characteristic similarities with the phosphate transporters of Neurospora crassa. The Pho84 protein catalyzes a proton-coupled phosphate transport at acidic pH, while the Pho89 protein catalyzes a sodium-dependent phosphate uptake at alkaline pH. The Pho84 transporter can be stably overproduced in the cytoplasmic membrane of Escherichia coli, purified and reconstituted in a functional state into proteoliposomes.

Structural modeling of dual-affinity purified Pho84 phosphate transporter

The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three‐dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol‐3‐phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template‐generated model, thus supporting the model.

Proton- and Sodium-Coupled Phosphate transport Systems and Energy Status of Yarrowia lipolytica Cells Grown in Acidic and Alkaline Conditions

Abstract. 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.

Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H+ transceptor and its effect on signalling to the PKA and PHO pathways

In Saccharomyces cerevisiae, the Pho84 phosphate transporter acts as the main provider of phosphate to the cell using a proton symport mechanism, but also mediates rapid activation of the PKA (protein kinase A) pathway. These two features led to recognition of Pho84 as a transceptor. Although the physiological role of Pho84 has been studied in depth, the mechanisms underlying the transport and sensor functions are unclear. To obtain more insight into the structure-function relationships of Pho84, we have rationally designed and analysed site-directed mutants. Using a three-dimensional model of Pho84 created on the basis of the GlpT permease, complemented with multiple sequence alignments, we selected Arg(168) and Lys(492), and Asp(178), Asp(358) and Glu(473) as residues potentially involved in phosphate or proton binding respectively, during transport. We found that Asp(358) (helix 7) and Lys(492) (helix 11) are critical for the transport function, and might be part of the putative substrate-binding pocket of Pho84. Moreover, we show that alleles mutated in the putative proton-binding site Asp(358) are still capable of strongly activating PKA pathway targets, despite their severely reduced transport activity. This indicates that signalling does not require transport and suggests that mutagenesis of amino acid residues involved in binding of the co-transported ion may constitute a promising general approach to separate the transport and signalling functions in transceptors.

Isolation and Characterization of a Novel Leaf-Inhabiting Osmo-, Salt-, and Alkali-Tolerant Yarrowia lipolytica Yeast Strain

Salt‐excreting leaves of Atriplex halimus plants harvested in the central Negev Highlands of Israel were screened for yeasts inhabiting their surfaces. Several aerobic, moderately salt‐ and alkalitolerant yeasts were isolated. One of the isolates (tentatively designated S‐8) was identified as Yarrowia lipolytica (Wick.) van der Walt and Arx, on the basis of its morphological, biochemical/physiological characteristics, and of quantitative chemotaxonomic and molecular marker analyses. However, the strain is distinguished from the known members of the type Y. lipolytica strain by its pronounced osmo‐, salt‐, and pH tolerance. Cells displayed a unique capacity to grow over a wide pH range (from 3.5 to 11.5) with a pH optimum at 4.5 to 7.5. It is proposed that the S‐8 strain be assigned to a single Y. lipolytica species as its anamorpha, or as a new variety, Y. lipolytica var. alkalitolerance. The ecophysiological properties and biotechnological potentials of the new strain are discussed.

Energy-linked nicotinamide nucleotide transhydrogenase: Hydrodynamic properties and active form of purified and membrane-bound mitochondrial transhydrogenase from beef heart

The mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to minimal assembly of the purified enzyme and of the enzyme in the mitochondrial inner membrane. Studies of the hydrodynamic properties of the purified enzyme in the presence of 0.3% Triton X-100 allowed determination of the Stokes radius, sedimentation constant, partial specific volume, frictional ratio, and molecular weight. Under these conditions transhydrogenase existed as an inactive monomer, suggesting that monomerization may be accompanied by inactivation. Radiation inactivation was used to determine the functional molecular size of purified detergent-dispersed transhydrogenase and transhydrogenase in beef heart submitochondrial particles. Under these conditions the catalytic activity of both the purified and the membrane-bound enzyme was found to be catalyzed by a dimeric form of the enzyme. These results suggest for the first time that the minimal functional assembly of detergent-dispersed as well as membrane-bound transhydrogenase is a dimer, which is not functionally associated with, for example, complex I or ATPase. In addition, the results are consistent with the possibility that the two subunits of transhydrogenase are catalytically active in an alternating fashion according to a previously proposed half-of-the-sites reactivity model.

Inhibition of the protein kinase A alters the degradation of the high-affinity phosphate transporter Pho84 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, nutrient sensing is the major factor controlling cell growth and proliferation. It has been shown that phosphate signalling involves the activation of the protein kinase A (PKA) in response to an elevation of external phosphate when cells have experienced a severe phosphate limitation. Addition of phosphate or its non-metabolized analogue, methylphosphonate (MP), to cells grown under phosphate limitation triggers degradation of the Pho84 phosphate transporter and represses the acidic phosphatase activity. In this study we have shown that of the five inorganic phosphate transporters (Pho84, Pho87, Pho89, Pho90, Pho91) of the plasma membrane, only Pho84 is required for the MP recognition and repression of the acidic phosphatase activity. By use of the PKA inhibitor H89, we demonstrate that down-regulation and degradation of the Pho84 transporter, in response to an elevation of external phosphate, is delayed by the inhibition of PKA. In contrast, down-regulation of the acidic phosphatase is under these conditions not affected by the PKA inhibition. Altogether, these observations suggest that the PKA signalling pathway plays a role in conveying the signal for the down-regulation and degradation of the Pho84 transporter in the vacuolar compartment in response to altered phosphate availability in the external environment.