Immunity to K1 killer toxin: internal TOK1 blockade

K1 killer strains of Saccharomyces cerevisiae harbor RNA viruses that mediate secretion of K1, a protein toxin that kills virus-free cells. Recently, external K1 toxin was shown to directly activate TOK1 channels in the plasma membranes of sensitive yeast cells, leading to excess potassium flux and cell death. Here, a mechanism by which killer cells resist their own toxin is shown: internal toxin inhibits TOK1 channels and suppresses activation by external toxin.     

Splitting the two pore domains from TOK1 results in two cationic channels with novel functional properties.

Potassium channels are membrane-spanning proteins with several transmembrane segments and a single pore region where ion conduction takes place (Biggin, P. C., Roosild, T., and Choe, S. (2000) Curr. Opin. Struct. Biol. 4, 456-461; Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77). TOK1, a potassium channel identified in the yeast Saccharomyces cerevisiae, was the first described member from a growing new family of potassium channels with two pore domains in tandem (2P) (Ketchum, K. A., Joiner, W. J., Sellers, A. J., Kaczmarek, L. K., and Goldstein, S. A. (1995) Nature 376, 690-695). In an attempt to understand the relative contribution of each one of the 2P from TOK1 to the functional properties of this channel, we split and expressed the pore domains separately or in combination. Expression of the two domains separately rescued a potassium transport-deficient yeast mutant, suggesting that each domain forms functional potassium-permeable channels in yeast. In Xenopus laevis oocytes expression of each pore domain resulted in the appearance of unique inwardly rectifying cationic channels with novel gating and pharmacological properties. Both pore domains were poorly selective to potassium; however, upon co-expression they partially restored TOK1 channel selectivity. The single channel conductance was different in both pore domains with 7 +/- 1 (n = 12) and 15 +/- 2 (n = 12) picosiemens for the first and second domain, respectively. In light of the known structure of the Streptomyces lividans KcsA potassium channel pore (see Doyle et al. above), these results suggest a novel non-four-fold-symmetric architecture for 2P potassium-selective channels.     

Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance

K+ is one of the cations (besides protons) whose transport across the plasma membrane is believed to contribute to the maintenance of membrane potential. To ensure K+ transport, Saccharomyces cerevisiae cells possess several types of active and passive transporters mediating the K+ influx and efflux, respectively. A diS-C3(3) assay was used to compare the contributions of various potassium transporters to the membrane potential changes of S. cerevisiae cells in the exponential growth phase. Altogether, the contributions of six K+ transporters to the maintenance of a stable membrane potential were tested. As confirmed by the observed hyperpolarization of trk1 trk2 deletion strains, the diS-C3(3) assay is a suitable method for comparative studies of the membrane potential of yeast strains differing in the presence/absence of one or more cation transporters. We have shown that the presence of the Tok1 channel strongly influences membrane potential: deletion of the TOK1 gene results in significant plasma membrane depolarization, whereas strains overexpressing the TOK1 gene are hyperpolarized. We have also proved that plasma membrane potential is not the only parameter determining the hygromycin B sensitivity of yeast cells, and that the role of intracellular transporters in protecting against its toxic effects must also be considered.     

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.     

Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.     

Antimicrobial action of palmarosa oil (Cymbopogon martinii) on Saccharomyces cerevisiae

The essential oil extracted from palmarosa (Cymbopogon martinii) has proven anti-microbial properties against cells of Saccharomyces cerevisiae. Low concentrations of the oil (0.1%) inhibited the growth of S. cerevisiae cells completely. The composition of the sample of palmarosa oil was determined as 65% geraniol and 20% geranyl acetate as confirmed by GC-FTIR. The effect of palmarosa oil in causing K(+) leakage from yeast cells was attributed mainly to geraniol. Some leakage of magnesium ions was also observed. Blocking potassium membrane channels with caesium ions before addition of palmarosa oil did not change the extent of K(+) ion leakage, which was equal to the total sequestered K(+) in the cells. Palmarosa oil led to changes in the composition of the yeast cell membrane, with more saturated and less unsaturated fatty acids in the membrane after exposure of S. cerevisiae cells to the oil. Some of the palmarosa oil was lost by volatilization during incubation of the oil with the yeast cells. The actual concentration of the oil components affecting the yeast cells could not therefore be accurately determined.     

Functional expression of the voltage-gated neuronal mammalian potassium channel rat ether à go-go1 in yeast.

It has been shown previously that heterologous expression of inwardly rectifying potassium channels (K+-channels) from plants and mammals in K+-transport defective yeast mutants can restore the ability of growth in media with low [K+]. In this study, the functional expression of an outward rectifying mammalian K+-channel in yeast is presented for the first time. The outward-rectifying mammalian neuronal K+-channel rat ether à go-go channel 1 (rEAG1, Kv 10.1) was expressed in yeast (Saccharomyces cerevisiae) strains lacking the endogenous K+-uptake systems and/or alkali-metal-cation efflux systems. It was found that a truncated channel version, lacking almost the complete intracellular N-terminus (rEAG1 Delta 190) but not the full-length rEAG1, partially complemented the growth defect of K+-uptake mutant cells (trk1,2 Delta tok1 Delta) in media containing low K+ concentrations. The expression of rEAG1 Delta 190 in a strain lacking the cation efflux systems (nha1 Delta ena1-4 Delta) increased the sensitivity to high monovalent cation concentrations. Both phenotypes were observed, when rEAG1 Delta 190 was expressed in a trk1,2 Delta and nha1, ena1-4 Delta mutant strain. In the presence of K+-channel blockers (Cs+, Ba2+ and quinidine), the growth advantage of rEAG1 Delta 190 expressing trk1,2 tok1 Delta cells disappeared, indicating its dependence on functional rEAG1 channels. The results demonstrate that S. cerevisiae is a suitable expression system even for voltage-gated outward-rectifying mammalian K+-channels.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Expression of pGKL killer 28K subunit in Saccharomyces cerevisiae: identification of 28K subunit as a killer protein.

Saccharomyces cerevisiae and other yeast cells harboring the linear double stranded (ds) DNA plasmids pGKL1 and pGKL2 secrete a killer toxin consisting of 97K, 31K and 28K subunits into the culture medium (EMBO J. 5, 1995-2002 (1986), Nucleic Acids Res., 15, 1031-1046 (1987]. The 28K subunit of the killer toxin was successfully expressed in S. cerevisiae when it was cloned on a circular plasmid with its putative promoter region replaced with that of S. cerevisiae chromosomal genes. The expression of the 28K subunit of the killer toxin in killer-sensitive cells resulted in the death of the host cells. This killing activity by the 28K subunit was prevented by the expression of the killer immunity, indicating that the killing activity of the killer toxin complex was carried out by the 28K subunit. Although the 28K subunit was synthesized as a intact precursor protein with its own signal sequence, it was not secreted into the culture medium but remained in the host cells. This indicated that 28K subunit killed host cells from inside of the cells rather than from outside. We further suggested that 28K killer subunit without 97K and 31K subunits did not kill the killer-sensitive cells from outside.     

Secrets of the fungus-specific potassium channel TOK family

Several families of potassium (K⁺) channels are found in membranes of all eukaryotes, underlining the importance of K⁺ uptake and redistribution within and between cells and organs. Among them, TOK (tandem-pore outward-rectifying K⁺) channels consist of eight transmembrane domains and two pore domains per subunit organized in dimers. These channels were originally studied in yeast, but recent identifications and characterizations in filamentous fungi shed new light on this fungus-specific K⁺ channel family. Although their actual function in vivo is often puzzling, recent works indicate a role in cellular K⁺ homeostasis and even suggest a role in plant–fungus symbioses. This review aims at synthesizing the current knowledge on fungal TOK channels and discussing their potential role in yeasts and filamentous fungi.     

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.     

Potassium Uptake Through the TOK1 K+ Channel in the Budding Yeast

The current through TOK1 (YKC1), the outward-rectifying K⁺ channel in Saccharomyces cerevisiae, was amplified by expressing TOK1 from a plasmid driven by a strong constitutive promoter. TOK1 so hyper-expressed could overcome the K⁺ auxotrophy of a mutant missing the two K⁺ transporters, TRK1 and TRK2. This trk1Δtrk2Δ double mutant hyperexpressing the TOK1 transgene had a higher internal K⁺ content than one expressing the empty plasmid. We examined protoplasts of these TOK1-hyperexpressing cells under a patch clamp. Besides the expected K⁺ outward current activating at membrane potential (V _m ) above the K⁺ equilibrium potential (E _K+ ), a small inward current was consistently observed when the V _m was slightly below E _K+ . The inward and the outward currents are similar in their activation rates, deactivation rates, ion specificities and Ba²⁺ inhibition, indicating that they flow through the same channel. Thus, the yeast outwardly rectifying K⁺ channel can take up K⁺ into yeast cells, at least under certain conditions. Received: 1 October 1998/Revised: 9 December 1998     

K(+)-dependent composite gating of the yeast K(+) channel, Tok1

TOK1 encodes an outwardly rectifying K(+) channel in the plasma membrane of the budding yeast Saccharomyces cerevisiae. It is capable of dwelling in two kinetically distinct impermeable states, a near-instantaneously activating R state and a set of related delayed activating C states (formerly called C(2) and C(1), respectively). Dwell in the R state is dependent on membrane potential and both internal and external K(+) in a manner consistent with the K(+) electrochemical potential being its determinant, where dwell in the C states is dependent on voltage and only external K(+). Whereas activation from the C states showed high temperature dependencies, typical of gating transitions in other Shaker-like channels, activation from the R state had a temperature dependence nearly as low as that of simple ionic diffusion. These findings lead us to conclude that although the C states reflect the activity of an internally oriented channel gate, the R state results from an intrinsic gating property of the channel filter region.     

Mode of action of yeast toxins: energy requirement for Saccharomyces cerevisiae killer toxin.

The role of the energy status of the yeast cell in the sensitivity of cultures to two yeast toxins was examined by using 12K release from cells as a measure of toxin action. The Saccharomyces cerevisiae killer toxin bound to sensitive cells in the presence of drugs that interfered with the generation or use of energy, but it was unable to efflux 12K from the cells under these conditions. In direct contrast, the Torulopsis glabrata pool efflux-stimulating toxin induced efflux of the yeast 42K pool was insensitive to the presence of energy poisons in cultures. The results indicate that an energized state, maintained at the expense of adenosine 5'-triphosphate from either glycolytic or mitochondrial reactions, is required for the action of the killer toxin on the yeast cell.     

Isolation, purification, and characterization of a killer protein from Schwanniomyces occidentalis

The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. The protein was stable between pH 2.0 and 5.0 and inactivated at temperatures above 40 degrees C. The killer protein was chromosomally encoded. Mannan, but not beta-glucan or laminarin, prevented sensitive yeast cells from being killed by the killer protein, suggesting that mannan may bind to the killer protein. Identification and characterization of a killer strain of S. occidentalis may help reduce the risk of contamination by undesirable yeast strains during commercial fermentations.     

Some properties of the adenosine triphosphatase systems of two yeast species,saccharomyces cerevisiae and rhodotorula glutinis

1. Total ATPase levels were determined in homogenate fractions of baker's yeast, Saccharomyces cerevisiae K and Rhodotorula glutinis. The maximum ATPase activities in 8000 X g supernatant of the three yeast strains were 6.0, 1.9, and 2.2 mmol Pih-1 (gDS)-1, respectively; the activities in the sediment were somewhat higher. Exponential cells of S. cerevisiae K and R. glutinis exhibited higher ATPase levels than did the stationary cells. 2. The total ATPase activity in both yeast species showed a maximum at ph 6.8 a minimum at pH 7.2, and another broader masimum around pH 8.0. 3. No significant NaK-ATPase activity was detected in baker's yeast, in either the exponential or the stationary cells of R. glutinis, and in exponential S. cerevisiae K cells in the pH range of 6.0-9.3. 4. Stationary cells of S. cerevisiae K exhibited, at pH 7.0-8.5, A Na,K-ATPase activity attaining 9% of total ATPase level. 5.3 X 10(-3) M phenylmethyl sulphonyl fluoride had no effect on the total ATPase level in S. cerevisiae and inhibited the activity in R. glutinis by 25%; it did not bring forth any Na,K-ATPase activity apart from that found in its absence. 6. 1.5 M urea lowered the ATPase activity in R. glutinis by 68% but had no effect on S. cerevisiae cells. 10(-5) M dicyclohexylcarbodiimide suppressed the ATPase activity in S. cerevisiae and R. glutinis by 74 and 79%, respectively. Neither agent revealed and additional Na,K-ATPase activity. 7. The comparison of Na,K-ATPase activities with data on K+ fluxes across the yeast plasma membrane suggested that even with the lower flux values the Na,K-ATPase, even if present, would account for a mere 40% of transported ions. The results imply that the active ion transport in yeasts is energized by mechanisms other than the Na,K-ATPase.     

Secretion of Saccharomyces cerevisiae Killer Toxin: Processing of the Glycosylated Precursor

Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In sec18 the protoxin was stable after a chase; but in sec7 and sec1 the protoxin was unstable, and in sec1 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and sec1 cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kex1 but unstable in kex2. Protoxin stability in kex1 kex2 double mutants indicated the order kex1 --> kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEX1- and KEX2-dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.