Chloride Channel Function in the Yeast TRK-Potassium Transporters

The TRK proteins—Trk1p and Trk2p— are the main agents responsible for “active” accumulation of potassium by the yeast Saccharomyces cerevisiae. In previous studies, inward currents measured through those proteins by whole-cell patch-clamping proved very unresponsive to changes of extracellular potassium concentration, although they did increase with extracellular proton concentration—qualitatively as expected for H⁺ coupling to K⁺ uptake. These puzzling observations have now been explored in greater detail, with the following major findings: a) the large inward TRK currents are not carried by influx of either K⁺ or H⁺, but rather by an efflux of chloride ions; b) with normal expression levels for Trk1p and Trk2p in potassium-replete cells, the inward TRK currents are contributed approximately half by Trk1p and half by Trk2p; but c) strain background strongly influences the absolute magnitude of these currents, which are nearly twice as large in W303-derived spheroplasts as in S288c-derived cells (same cell-size and identical recording conditions); d) incorporation of mutations that increase cell size (deletion of the Golgi calcium pump, Pmr1p) or that upregulate the TRK2 promoter, can further substantially increase the TRK currents; e) removal of intracellular chloride (e.g., replacement by sulfate or gluconate) reveals small inward currents that are K⁺-dependent and can be enhanced by K⁺ starvation; and f) finally, the latter currents display two saturating kinetic components, with preliminary estimates of K_0.5 at 46 μM [K⁺]_out and 6.8 mM [K⁺]_out, and saturating fluxes of ∼5 mM/min and ∼10 mM/min (referred to intracellular water). These numbers are compatible with the normal K⁺-transport properties of Trk1p and Trk2p, respectively.This revised version was published online in August 2005 with a corrected cover date.     

Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations

Saccharomyces cerevisiae cells express three defined potassium-specific transport systems en-coded by TRK1, TRK2 and TOK1. To gain a more complete understanding of the physiological function of these transport proteins, we have constructed a set of isogenic yeast strains carrying all combinations of trk1delta, trk2delta and tok1delta null mutations. The in vivo K+ transport characteristics of each strain have been documented using growth-based assays, and the in vitro biochemical and electrophysiological properties associated with K+ transport have been determined. As has been reported previously, Trk1p and Trk2p facilitate high-affinity potassium uptake and appear to be functionally redundant under a wide range of environmental conditions. In the absence of TRK1 and TRK2, strains lack the ability specifically to take up K+, and trk1deltatrk2delta double mutant cells depend upon poorly understood non-specific cation uptake mechanisms for growth. Under conditions that impair the activity of the non-specific uptake system, termed NSC1, we have found that the presence of functional Tok1p renders cells sensitive to Cs+. Based on this finding, we have established a growth-based assay that monitors the in vivo activity of Tok1p.     

A refined atomic scale model of the Saccharomyces cerevisiae K+-translocation protein Trk1p combined with experimental evidence confirms the role of selectivity filter glycines and other key residues

Potassium ion (K⁺) uptake in yeast is mediated mainly by the Trk1/2 proteins that enable cells to survive on external K⁺ concentration as low as a few μM. Fungal Trks are related to prokaryotic TRK and Ktr and plant HKT K⁺ transport systems. Overall sequence similarity is very low, thus requiring experimental verification of homology models. Here a refined structural model of the Saccharomyces cerevisiae Trk1 is presented that was obtained by combining homology modeling, molecular dynamics simulation and experimental verification through functional analysis of mutants. Structural models and experimental results showed that glycines within the selectivity filter, conserved among the K-channel/transporter family, are not only important for protein function, but are also required for correct folding/membrane targeting.A conserved aspartic acid in the P_A helix (D79) and a lysine in the M2_D helix (K1147) were proposed earlier to interact. Our results suggested individual roles of these residues in folding, structural integrity and function. While mutations of D79 completely abolished protein folding, mutations at position 1147 were tolerated to some extent. Intriguingly, a secondary interaction of D79 with R76 could enhance folding/stability of Trk1 and enable a fraction of Trk1[K1147A] to fold.The part of the ion permeation path containing the selectivity filter is shaped similar to that of ion channels. However below the selectivity filter it is obstructed or regulated by a proline containing loop. The presented model could provide the structural basis for addressing the long standing question if Trk1 is a passive or active ion-translocation system.     

Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions

Saccharomyces cerevisiae extrudes K(+) cations even when potassium is only present in scarce amounts in the environment. Lost potassium is taken up by the Trk1 and Trk2 uptake systems. If the Trk transporters are absent or nonfunctional, the efflux of potassium is significantly diminished. A series of experiments with strains lacking various combinations of potassium efflux and uptake systems revealed that all three potassium-exporting systems the Nha1 antiporter, Ena ATPase and Tok1 channel contribute to potassium homeostasis and are active upon potassium limitation in wild-type cells. In trk1Δ trk2Δ mutants, the potassium efflux via potassium exporters Nha1 and Ena1 is diminished and can be restored either by the expression of TRK1 or deletion of TOK1. In both cases, the relative hyperpolarization of trk1Δ trk2Δ cells is decreased. Thus, it is the plasma-membrane potential which serves as the common mechanism regulating the activity of K(+) exporting systems. There is a continuous uptake and efflux of potassium in yeast cells to regulate their membrane potential and thereby other physiological parameters, and the cells are able to quickly and efficiently compensate for a malfunction of potassium transport in one direction by diminishing the transport in the other direction.     

TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae.

We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion.     

Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions

Background

Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.

Results

Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.

Conclusions

Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.     

Quantitative modeling of chloride conductance in yeast TRK potassium transporters

So-called TRK proteins are responsible for active accumulation of potassium in plants, fungi, and bacteria. A pair of these proteins in the plasma membrane of Saccharomyces cerevisiae, ScTrk1p and ScTrk2p, also admit large, adventitious, chloride currents during patch-recording (Cl- efflux). Resulting steady-state current-voltage curves can be described by two simple kinetic models, most interestingly, voltage-driven channeling of ions through a pair of activation-energy barriers that lie within the membrane dielectric, near the inner (alpha) and outer (beta) surfaces. Two barrier heights (E(alpha) and E(beta)) and two relative distances (a1 and b2) from the surfaces specify the model. Measured current amplitude parallels intracellular chloride concentration and is strongly enhanced by acidic extracellular pH. The former implies an exponential variation of a1, between approximately 0.2 and approximately 0.4 of the membrane thickness, whereas the latter implies a linear variation of E(beta), by 0.69 Kcal mol(-1)/pH. The model requires membrane slope conductance to rise exponentially with increasingly large negative membrane voltage, as verified by data from a few yeast spheroplasts that tolerated voltage clamping at -200 to -300 mV. The behaviors of E(beta) and a1 accord qualitatively with a hypothetical structural model for fungal TRK proteins, suggesting that chloride ions flow through a central pore formed by symmetric aggregation of four TRK monomers.     

The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5

The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K(+), which can be blocked by the anion channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organism's principal K(+) transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K(+) ((86)Rb(+)) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5-7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.     

Prokaryotic Ubiquitin-Like ThiS Fusion Enhances the Heterologous Protein Overexpression and Aggregation in Escherichia coli

Fusion tags are commonly employed to enhance target protein expression, improve their folding and solubility, and reduce protein degradation in expression of recombinant proteins. Ubiquitin (Ub) and SUMO are highly conserved small proteins in eukaryotes, and frequently used as fusion tags in prokaryotic expression. ThiS, a smaller sulfur-carrier protein involved in thiamin synthesis, is conserved among most prokaryotic species. The structural similarity between ThiS and Ub provoked us into expecting that the former could be used as a fusion tag. Hence, ThiS was fused to insulin A and B chains, murine Ribonuclease Inhibitor (mRI) and EGFP, respectively. When induced in Escherichia coli, ThiS-fused insulin A and B chains were overexpressed in inclusion bodies, and to higher levels in comparison to the same proteins fused with Ub. On the contrast, ThiS fusion of mRI, an unstable protein, resulted in enhanced degradation that was not alleviated in protease-deficient strains. While the degradation of Ub- and SUMO-fused mRI was less and seemed protease-dependent. Enhanced degradation of mRI did not occur for the fusions with half-molecules of ThiS. When ThiS-tag was fused to the C-terminus of EGFP, higher expression, predominantly in inclusion bodies, was observed again. It was further found that ThiS fusion of EGFP significantly retarded its refolding process. These results indicated that prokaryotic ThiS is able to promote the expression of target proteins in E. coli, but enhanced degradation may occur in case of unstable targets. Unlike eukaryotic Ub-based tags usually increase the solubility and folding of proteins, ThiS fusion enhances the expression by augmenting the formation of inclusion bodies, probably through retardation of the folding of target proteins.     

Topology of the yeast fatty acid transport protein Fat1p: mechanistic implications for functional domains on the cytosolic surface of the plasma membrane

The fatty acid transport protein (FATP) Fat1p in the yeast Saccharomyces cerevisiae functions in concert with acyl-coenzyme A synthetase (ACSL; either Faa1p or Faa4p) in vectorial acylation, which couples the transport of exogenous fatty acids with activation to CoA thioesters. To further define the role of Fat1p in the transport of exogenous fatty acids, the topological orientation of two highly conserved motifs [ATP/AMP and FATP/very long chain acyl CoA synthetase (VLACS)], the carboxyl 124 amino acid residues, which bind the ACSL Faa1p, and the amino and carboxyl termini within the plasma membrane were defined. T7 or hemagglutinin epitope tags were engineered at both amino and carboxyl termini, as well as at multiple nonconserved, predicted random coil segments within the protein. Six different epitope-tagged chimeras of Fat1p were generated and expressed in yeast; the sidedness of the tags was tested using indirect immunofluorescence and protease protection by Western blotting. Plasma membrane localization of the tagged proteins was assessed by immunofluorescence. Fat1p appears to have at least two transmembrane domains resulting in a N(in)-C(in) topology. We propose that Fat1p has a third region, which binds to the membrane and separates the highly conserved residues comprising the two halves of the ATP/AMP motif. The N(in)-C(in) topology results in the placement of the ATP/AMP and FATP/VLACS domains of Fat1p on the inner face of the plasma membrane. The carboxyl-terminal region of Fat1p, which interacts with ACSL, is likewise positioned on the inner face of the plasma membrane. This topological orientation is consistent with the mechanistic roles of both Fat1p and Faa1p or Faa4p in the coupled transport/activation of exogenous fatty acids by vectorial acylation.     

Improved Blue,Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae

Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging.     

Individual functions of the HAK and TRK potassium transporters of Schwanniomyces occidentalis

We have cloned the gene encoding the TRK transporter of the soil yeast Schwanniomyces occidentalis and obtained the HAK1 trk1 delta and the hak1 delta TRK1 mutant strains. Analyses of the transport capacities of these mutants have shown that (i) the HAK1 and the TRK1 potassium transporters are the only transporters operating at low and medium K+ concentrations (< 1 mM); (ii) the HAK1 transporter is functional at low pH but fails at high pH; and (iii) the TRK1 transporter functions at neutral and high pH and fails at low pH. At neutral pH, both transporters are functional, but HAK1 is not expressed, except at very low K+ concentrations (< 50 microM) where HAK1 is very effective. TRK1 is also involved in the control of the membrane potential.     

Kch1 Family Proteins Mediate Essential Responses to Endoplasmic Reticulum Stresses in the Yeasts Saccharomyces cerevisiae and Candida albicans

The activation of a high affinity Ca²⁺ influx system (HACS) in the plasma membrane is required for survival of yeast cells exposed to natural or synthetic inhibitors of essential processes (secretory protein folding or sterol biosynthesis) in the endoplasmic reticulum (ER). The mechanisms linking ER stress to HACS activation are not known. Here we show that Kch1, a recently identified low affinity K⁺ transporter in the plasma membrane of Saccharomyces cerevisiae, is up-regulated in response to several ER stressors and necessary for HACS activation. The activation of HACS required extracellular K⁺ and was also dependent on the high affinity K⁺ transporters Trk1 and Trk2. However, a paralog of Kch1 termed Kch2 was not expressed and not necessary for HACS activation in these conditions. The pathogenic yeast Candida albicans carries only one homolog of Kch1/Kch2, and homozygous knock-out mutants were similarly deficient in the activation of HACS during the responses to tunicamycin. However, the Kch1 homolog was not necessary for HACS activation or cell survival in response to several clinical antifungals (azoles, allylamines, echinocandins) that target the ER or cell wall. Thus, Kch1 family proteins represent a conserved linkage between HACS and only certain classes of ER stress in these yeasts.     

Trk1 and Trk2 define the major K(+) transport system in fission yeast

The trk1(+) gene has been proposed as a component of the K(+) influx system in the fission yeast Schizosaccharomyces pombe. Previous work from our laboratories revealed that trk1 mutants do not show significantly altered content or influx of K(+), although they are more sensitive to Na(+). Genome database searches revealed that S. pombe encodes a putative gene (designated here trk2(+)) that shows significant identity to trk1(+). We have analyzed the characteristics of potassium influx in S. pombe by using trk1 trk2 mutants. Unlike budding yeast, fission yeast displays a biphasic transport kinetics. trk2 mutants do not show altered K(+) transport and exhibit only a slightly reduced Na(+) tolerance. However, trk1 trk2 double mutants fail to grow at low K(+) concentrations and show a dramatic decrease in Rb(+) influx, as a result of loss of the high-affinity transport component. Furthermore, trk1 trk2 cells are very sensitive to Na(+), as would be expected for a strain showing defective potassium transport. When trk1 trk2 cells are maintained in K(+)-free medium, the potassium content remains higher than that of the wild type or trk single mutants. In addition, the trk1 trk2 strain displays increased sensitivity to hygromycin B. These results are consistent with a hyperpolarized state of the plasma membrane. An additional phenotype of cells lacking both Trk components is a failure to grow at acidic pH. In conclusion, the Trk1 and Trk2 proteins define the major K(+) transport system in fission yeast, and in contrast to what is known for budding yeast, the presence of any of these two proteins is sufficient to allow growth at normal potassium levels.     

Crystal structures of fusion proteins with large-affinity tags

The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.     

Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane

Yeast plasma membranes contain a small 55 amino acid hydrophobic polypeptide, Pmp3p, which has high sequence similarity to a novel family of plant polypeptides that are overexpressed under high salt concentration or low temperature treatment. The PMP3 gene is not essential under normal growth conditions. However, its deletion increases the plasma membrane potential and confers sensitivity to cytotoxic cations, such as Na(+) and hygromycin B. Interestingly, the disruption of PMP3 exacerbates the NaCl sensitivity phenotype of a mutant strain lacking the Pmr2p/Enap Na(+)-ATPases and the Nha1p Na(+)/H(+) antiporter, and suppresses the potassium dependency of a strain lacking the K(+) transporters, Trk1p and Trk2p. All these phenotypes could be reversed by the addition of high Ca(2+) concentration to the medium. These genetic interactions indicate that the major effect of the PMP3 deletion is a hyperpolarization of the plasma membrane potential that probably promotes a non-specific influx of monovalent cations. Expression of plant RCI2A in yeast could substitute for the loss of Pmp3p, indicating a common role for Pmp3p and the plant homologue.     

Fluorescent muscarinic EGFP-hM1 chimeric receptors: design, ligand binding and functional properties

We describe the construction, expression and characterization of recombinant proteins comprising the enhanced green fluorescent protein (EGFP) fused to the amino-terminal part of the muscarinic hM1 receptor together or not with an additional hexahistidine tag placed at the C-terminal end of the receptor. Expression of the fluorescent proteins reaches levels identical to those of the wt hM1 receptor, provided that fusion takes place at the very N-terminal end of the receptor. Also correct protein folding and targeting to plasma membrane is obtained upon addition of a signal peptide promoting amino-terminal domain translocation through the membrane. Ligand binding properties of--and activation of the calcium release response by--the fusion proteins are almost identical to those of the wild-type muscarinic receptor, indicating that such fluorescently-labelled receptors are valuable model systems for further functional, biochemical and structural studies.     

Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion‐to‐fusion transition

SNAREs fuse membranes in several steps. Trans‐SNARE complexes juxtapose membranes, induce hemifused stalk structures, and open the fusion pore. A recent penetration model of fusion proposed that SNAREs force the hydrophilic C‐termini of their transmembrane domains through the hydrophobic core of the membrane(s). In contrast, the indentation model suggests that the C‐termini open the pore by locally compressing and deforming the stalk. Here we test these models in the context of yeast vacuole fusion. Addition of small hydrophilic tags renders bilayer penetration by the C‐termini energetically unlikely. It preserves fusion activity, however, arguing against the penetration model. Addition of large protein tags to the C‐termini permits SNARE activation, trans‐SNARE pairing, and hemifusion but abolishes pore opening. Fusion proceeds if the tags are detached from the membrane by a hydrophilic spacer or if only one side of the trans‐SNARE complex carries a protein tag. Thus, both sides of a trans‐SNARE complex can drive pore opening. Our results are consistent with an indentation model in which multiple SNARE C‐termini cooperate in opening the fusion pore by locally deforming the inner leaflets.