Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae

The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na⁺/Li⁺-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li⁺-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li⁺ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li⁺-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to␣cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast. Received: 17 August 1998 / Accepted: 7 December 1998     

Regulation of Trk-dependent potassium transport by the calcineurin pathway involves the Hal5 kinase

The phosphatase calcineurin and the kinases Hal4/Hal5 regulate high-affinity potassium uptake in Saccharomyces cerevisiae through the Trk1 transporter. We demonstrate that calcineurin is necessary for high-affinity potassium uptake even in the absence of Na⁺ stress. HAL5 expression is induced in response to stress in a calcineurin-dependent manner through a newly identified functional CDRE (nt −195/−189). Lack of calcineurin decreases Hal5 protein levels, although with little effect on Trk1 amounts. However, the growth defect of cnb1 cells at K⁺-limiting conditions can be rescued in part by overexpression of HAL5, and this mutation further aggravates the potassium requirements of a hal4 strain. This suggests that the control exerted by calcineurin on Hal5 expression may be biologically relevant for Trk1 regulation.     

The yeast SR protein kinase Sky1p modulates salt tolerance, membrane potential and the Trk1,2 potassium transporter

Protein kinases dedicated to the phosphorylation of SR proteins have been implicated in the processing and nuclear export of mRNAs. Here we demonstrate in Saccharomyces cerevisiae their participation in cation homeostasis. A null mutant of the single yeast SR protein kinase Sky1p is viable but exhibits increased tolerance to diverse toxic cations such as Na⁺, Li⁺, spermine, tetramethylammonium, hygromycin B and Mn²⁺. This pleiotropic phenotype correlates with reduced accumulation of cations, suggesting a decrease in membrane electrical potential. Genetic analysis and Rb⁺ uptake measurements indicate that Sky1p modulates Trk1,2, the high-affinity K⁺ uptake system of yeast and a major determinant of membrane potential.     

Li+ effect on the cell wall of the yeast Saccharomyces cerevisiae as probed by FT-IR spectroscopy

The effect of Li⁺ ions as a transformation inducing agent on the yeast cell wall has been studied. Two Saccharomyces cerevisiae strains, p63-DC5 with a native cell wall, and strain XCY42-30D(mnn1) which contains structural changes in the mannan-protein complex, were used. Fourier transform infrared (FT-IR) spectroscopy has been used for the characterization of the yeast strains and for determination of the effect of lithium cations on the cell wall. A comparison of the carbohydrate absorption band positions in the 970–1185 cm^?1 range, of Na⁺ and Li⁺ treated yeast cells has been estimated. Absorption band positions of the cell wall carbohydrates of p63-DC5 were not influenced by the studied ions. On the contrary, the treatment of XCY42-30D(mnn1) cells with Li⁺ ions shifted glucan band positions, implying that the cell wall structure of strain XCY42-30D(mnn1) is more sensitive to Li⁺ ion treatment.     

Mutants of the Arabidopsis thaliana Cation/H+ Antiporter AtNHX1 Conferring Increased Salt Tolerance in Yeast: THE ENDOSOME/PREVACUOLAR COMPARTMENT IS A TARGET FOR SALT TOXICITY*

Mutants of the plant cation/H⁺ antiporter AtNHX1 that confer greater halotolerance were generated by random mutagenesis and selected in yeast by phenotypic complementation. The amino acid substitutions that were selected were conservative and occurred in the second half of the membrane-associated N terminus. AtNHX1 complemented the lack of endogenous ScNHX1 in endosomal protein trafficking assays. Growth enhancement on hygromycin B and vanadate media agreed with a generally improved endosomal/prevacuolar function of the mutated proteins. In vivo measurements by ³¹P NMR revealed that wild-type and mutant AtNHX1 transporters did not affect cytosolic or vacuolar pH. Surprisingly, when yeast cells were challenged with lithium, a tracer for sodium, the main effect of the mutations in AtNHX1 was a reduction in the amount of compartmentalized lithium. When purified and reconstituted into proteoliposomes or assayed in intact vacuoles isolated from yeast cells, a representative mutant transporter (V318I) showed a greater cation discrimination favoring potassium transport over that of sodium or lithium. Together, our data suggest that the endosome/prevacuolar compartment is a target for salt toxicity. Poisoning by toxic cations in the endosome/prevacuolar compartment is detrimental for cell functions, but it can be alleviated by improving the discrimination of transported alkali cations by the resident cation/H⁺ antiporter.The Arabidopsis thaliana vacuolar alkali cation transporter AtNHX1 has been shown to increase salt tolerance in transgenic plants of several species (1). In Saccharomyces cerevisiae, its ortholog (ScNHX1) is mainly localized in late endosomes, where it is thought to contribute to vacuole biogenesis by regulating pH and vesicle volume (2). ScNHX1 itself has a role in halotolerance. Deletion of ScNHX1 confers salt sensitivity and diminishes Na⁺ compartmentalization, albeit indirectly, since the unrelated VNX1 exchanger accounts for most of the cation/H⁺ antiport activity in the tonoplast of yeast (3, 4). However, AtNHX1 complements a yeast mutant defective in ScNHX1 and restores cation compartmentalization (5).Improving the salt tolerance of crop plants is an important goal in biotechnology. In addition to the mechanisms by which a cell can cope with increased concentrations of toxic cations, it is important to know the identity of salt-sensitive cellular targets. Only a few key processes have been identified. In yeast, HAL2, an inositol phosphatase that catalyzes the dephosphorylation of 3′-phosphoadenosine-5′-phosphate to AMP, has been found to be inhibited by Li⁺ and Na⁺. Inhibition of HAL2 during salt stress results in the accumulation of 3′-phosphoadenosine-5′-phosphate in the cell, which has the potential to produce a variety of toxic effects, such as the inhibition of sulfotransferases and RNA-processing enzymes (6). Another possible target is the KEX2/furin family of proteases of the Golgi/secretory pathway. The activity of KEX2 in vitro has been shown to respond differently, depending on the alkali cation and concentration present in the medium (7). Here, we show that the endosomal system is an additional target for Na⁺ toxicity.The Golgi apparatus, trans-Golgi network, and endosome/prevacuolar compartment form a continuum where proteins and membranes are modified en route to their final destinations (8–10). The late endosome/prevacuolar compartment is considered a key point in intracellular vesicle and protein trafficking. In addition to being the previous stage for vacuolar sorted proteins and cargo, this is where both the exocytic and endocytic pathways converge (10, 11). Ion homeostasis in these organelles is increasingly regarded as an important feature for intracellular transport processes (12–15). In particular, K⁺ concentration may regulate the activity and specificity of enzymes modifying proteins posttranslationally, such as the above mentioned KEX2/furin protease family (7). Lumenal pH has been reported also to regulate selective protein aggregation in secretory vesicles (12). In this respect, it is noteworthy that yeast nhx1 mutants have been characterized as class E vps mutants with impaired vacuole biogenesis and protein sorting (15).AtNHX1 is thought to increase salt tolerance in plants through the intracellular compartmentation of Na⁺. However, using purified protein, it has been shown that this antiporter can exchange H⁺ for K⁺, Na⁺, or Li⁺, albeit the last one with lower affinity (16). The poor K⁺/Na⁺ selectivity raises the question of whether Na⁺ transport is the primary function of AtNHX1 in plant cells and if AtNHX1 is amenable to selection of better alleles for salt tolerance. Mutagenesis of cation transporters has proved to be a valuable tool to obtain alleles with modified transport activities (17, 18). At the same time, this provides information about the important amino acid residues that affect the mechanism of protein function. In this work, we sought to produce hypermorphic AtNHX1 alleles conferring greater salt tolerance, by either improved Na⁺/K⁺ discrimination or altered protein regulation. We show here that nhx1-deficient yeast cells that express mutated forms of AtNHX1 display improved halotolerance compared with cells that express the wild-type AtNHX1. The mutations responsible for these changes were scattered throughout the hydrophobic N terminus of the protein, and their effect was to introduce bulkier side chain amino acids. Surprisingly, the result of these mutations was not increased compartmentalization of toxic alkali cations. Instead, all of these mutants showed a decreased content of Li⁺ (a tracer for Na⁺), whereas full amounts of K⁺ were retained. Biochemical characterization of a selected mutant transporter showed greater cation discrimination favoring K⁺ transport. AtNHX1 is localized to the vacuole and late endosome/prevacuolar compartment. Together, these results suggest that the endomembrane system is a cellular target of Na⁺ intoxication.     

Vhc1, a novel transporter belonging to the family of electroneutral cation–Cl− cotransporters,participates in the regulation of cation content and morphology of Saccharomyces cerevisiae vacuoles

Cation–Cl^? cotransporters (CCCs) are integral membrane proteins which catalyze the coordinated symport of Cl^? with Na⁺ and/or K⁺ ions in plant and mammalian cells. Here we describe the first Saccharomyces cerevisiae CCC protein, encoded by the YBR235w open reading frame. Subcellular localization studies showed that this yeast CCC is targeted to the vacuolar membrane. Deletion of the YBR235w gene in a salt-sensitive strain (lacking the plasma-membrane cation exporters) resulted in an increased sensitivity to high KCl, altered vacuolar morphology control and decreased survival upon hyperosmotic shock. In addition, deletion of the YBR235w gene in a mutant strain deficient in K⁺ uptake produced a significant growth advantage over the parental strain under K⁺-limiting conditions, and a hypersensitivity to the exogenous K⁺/H⁺ exchanger nigericin. These results strongly suggest that we have identified a novel yeast vacuolar ion transporter mediating a K⁺–Cl^? cotransport and playing a role in vacuolar osmoregulation. Considering its identified function, we propose to refer to the yeast YBR235w gene as VHC1 (vacuolar protein homologous to CCC family 1).     

Cation diffusion facilitator Cis4 is implicated in Golgi membrane trafficking via regulating zinc homeostasis in fission yeast

We screened for mutations that confer sensitivities to the calcineurin inhibitor FK506 and to a high concentration of MgCl₂ and isolated the cis4-1 mutant, an allele of the gene encoding a cation diffusion facilitator (CDF) protein that is structurally related to zinc transporters. Consistently, the addition of extracellular Zn²⁺ suppressed the phenotypes of the cis4 mutant cells. The cis4 mutants and the mutant cells of another CDF-encoding gene SPBC16E9.14c (we named zrg17⁺) shared common and nonadditive zinc-suppressible phenotypes, and Cis4 and Zrg17 physically interacted. Cis4 localized at the cis-Golgi, suggesting that Cis4 is responsible for Zn²⁺ uptake to the cis-Golgi. The cis4 mutant cells showed phenotypes such as weak cell wall and decreased acid phosphatase secretion that are thought to be resulting from impaired membrane trafficking. In addition, the cis4 deletion cells showed synthetic growth defects with all the four membrane-trafficking mutants tested, namely ypt3-i5, ryh1-i6, gdi1-i11, and apm1-1. Interestingly, the addition of extracellular Zn²⁺ significantly suppressed the phenotypes of the ypt3-i5 and apm1-1 mutant cells. These results suggest that Cis4 forms a heteromeric functional complex with Zrg17 and that Cis4 is implicated in Golgi membrane trafficking through the regulation of zinc homeostasis in fission yeast.     

Tandem repeats of the tetramer 5'-CAAT-3'present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae

A novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase-variable LPS structure expressed by Haemo philus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neis seria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)ⁿ encoded by multiple tandem repeats of 5′-CAAT-3′. This observation suggested that (SINQ)ⁿ might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5′-CAAT-3’repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine-SDS-PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)ⁿ is not a prerequisite for the enzymatic function of Lic2A and that the 5′-CAAT-3’repeats in lic2A function solely as a mechan ism for generating phase variation. This observation suggested that wide variation in the number of 5’-CAAT-3’repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5′-CAAT-3’repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)ⁿ indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.     

The Second Sodium Site in the Dopamine Transporter Controls Cation Permeation and Is Regulated by Chloride

The dopamine transporter (DAT) belongs to the family of neurotransmitter:sodium symporters and controls dopamine (DA) homeostasis by mediating Na⁺- and Cl⁻-dependent reuptake of DA. Here we used two-electrode voltage clamp measurements in Xenopus oocytes together with targeted mutagenesis to investigate the mechanistic relationship between DAT ion binding sites and transporter conductances. In Li⁺, DAT displayed a cocaine-sensitive cation leak current ∼10-fold larger than the substrate-induced current in Na⁺. Mutation of Na⁺ coordinating residues in the first (Na1) and second (Na2) binding sites suggested that the Li⁺ leak depends on Li⁺ interaction with Na2 rather than Na1. DA caused a marked inhibition of the Li⁺ leak, consistent with the ability of the substrate to interact with the Li⁺-occupied state of the transporter. The leak current in Li⁺ was also potently inhibited by low millimolar concentrations of Na⁺, which according to our mutational data conceivably depended on high affinity binding to Na1. The Li⁺ leak was further regulated by Cl⁻ that most likely increases Li⁺ permeation by allosterically lowering Na2 affinity. Interestingly, mutational lowering of Na2 affinity by substituting Asp-420 with asparagine dramatically increased cation permeability in Na⁺ to a level higher than seen in Li⁺. In addition to reveal a functional link between the bound Cl⁻ and the cation bound in the Na2 site, the data support a key role of Na2 in determining cation permeability of the transporter and thereby possibly in regulating the opening probability of the inner gate.     

Na+ (Li+)-proline cotransport inEscherichia coli

Summary Na⁺ and Li⁺ were found to stimulate the transport ofl-proline by cells ofEscherichia coli induced for proline utilization. The gene product of the put P gene is involved in the expression of this transport activity since the put P⁺ strains CSH 4 and WG 148 show activity and the put P^– strain RM 2 fails to show this cation coupled transport. The addition of proline was found to stimulate the uptake of Li⁺ and of Na⁺. Attempts to demonstrate proline stimulated H⁺ uptake were unsuccessful. It is concluded that the proline carrier (coded by the put P gene) is responsible for Na⁺ (or Li⁺)-proline cotransport.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K⁺, Na⁺, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Ni²⁺, Zn²⁺, and Li⁺) were analyzed. AtCCX5 expression was found to affect the response to K⁺ and Na⁺ in yeast. The AtCCX5 transformant also showed a little better growth to Zn²⁺. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K⁺ (0.5 mM), and also suppressed its Na⁺ sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K⁺ uptake and was also involved in Na⁺ transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K⁺ uptake and Na⁺ transport in yeast.     

Matrix-dependent modulation of anisotropic effects on NMR spectra from 7Li+ and 23Na+ encapsulated in cryptands

⁷Li and ²³Na NMR spectra of the respective cations in gelatin and ι-carrageenan gels containing cryptand-[2.1.1] (for Li⁺) or cryptand-[2.2.2] (for Na⁺) displayed two transitions: the one at higher frequency corresponded to the cation surrounded by gel, the other to cation inside its appropriately sized cryptand. While binding to cryptands yielded much broader lines and shorter T ₁ relaxation times, anisotropic splitting in first order ⁷Li or ²³Na NMR spectra was not detected. Stretching the gels resulted in increasing the anisotropic electric field gradient tensor; thus, the NMR transitions of the cation in the gel were split (removal of degeneracy) to display its characteristic 3:4:3 triplet for spin = 3/2 nuclei. The transitions of the cryptand-bound cations (Li⁺-cryptand-[2.1.1] and Na⁺-cryptand-[2.2.2]) showed different extents of interaction with the electric field gradient tensor depending on the composition of the gel matrix. The NMR signal for ⁷Li⁺-cryptand-[2.1.1] in stretched gelatin gel showed a five-fold increased splitting as compared to the ⁷Li⁺ signal in the reference gel. In stretched ι-carrageenan gels, no anisotropic splitting from the cryptand-bound Li⁺ was recorded. Steady-state irradiation envelopes or z-spectra showed evidence of Li⁺ exchange between isotropic (cryptand) and anisotropic (gel) sites only at higher temperatures (55 °C). For Na⁺ bound to the cryptand-[2.2.2], anisotropic splitting (three-fold smaller compared with the ²³Na signal in the reference gel) was only recorded in stretched ι-carrageenan gels, whereas gelatin gels showed only anisotropic splitting for the ²³Na signal in the reference gel.     

A Novel AtKEA Gene Family,Homolog of Bacterial K+/H+ Antiporters,Plays Potential Roles in K+ Homeostasis and Osmotic Adjustment in Arabidopsis

AtKEAs, homologs of bacterial KefB/KefC, are predicted to encode K⁺/H⁺ antiporters in Arabidopsis. The AtKEA family contains six genes forming two subgroups in the cladogram: AtKEA1-3 and AtKEA4-6. AtKEA1 and AtKEA2 have a long N-terminal domain; the full-length AtKEA1 was inactive in yeast. The transport activity was analyzed by expressing the AtKEA genes in yeast mutants lacking multiple ion carriers. AtKEAs conferred resistance to high K⁺ and hygromycin B but not to salt and Li⁺ stress. AtKEAs expressed in both the shoot and root of Arabidopsis. The expression of AtKEA1, -3 and -4 was enhanced under low K⁺ stress, whereas AtKEA2 and AtKEA5 were induced by sorbitol and ABA treatments. However, osmotic induction of AtKEA2 and AtKEA5 was not observed in aba2-3 mutants, suggesting an ABA regulated mechanism for their osmotic response. AtKEAs’ expression may not be regulated by the SOS pathway since their expression was not affected in sos mutants. The GFP tagging analysis showed that AtKEAs distributed diversely in yeast. The Golgi localization of AtKEA3 was demonstrated by both the stably transformed seedlings and the transient expression in protoplasts. Overall, AtKEAs expressed and localized diversely, and may play roles in K⁺ homeostasis and osmotic adjustment in Arabidopsis.     

Phospholipid-binding protein Cts1 controls septation and functions coordinately with calcineurin in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised patients. The Ca²⁺-calmodulin-activated protein phosphatase calcineurin is necessary for virulence of C. neoformans. Mutants lacking the calcineurin catalytic (Cna1) or regulatory (Cnb1) subunit fail to grow at elevated temperature and are defective in virulence and hyphal elongation. Here we isolated a multicopy suppressor gene, CTS1, which restores growth of a calcineurin mutant strain at 37°C. The CTS1 gene (for calcineurin temperature suppressor 1) encodes a protein containing a C2 domain and a leucine zipper motif that may function as an effector of calcineurin. The CTS1 gene was disrupted by homologous recombination, and cts1 mutants were viable but exhibited defects in cell separation, growth, mating, and haploid fruiting. In addition, cts1 mutants were inviable when calcineurin was mutated or inhibited. Taken together, these findings suggest that calcineurin and Cts1 function in parallel pathways that regulate growth, cell separation, and hyphal elongation.     

Osmotic swelling of heart mitochondria in acetate and chloride salts. Evidence for two pathways for cation uptake.

Swelling of nonenergized heart mitochondria suspended in acetate salts appears to depend on the activity of an endogenous cation/H⁺ exchanger. Passive swelling in acetate shows a characteristic cation selectivity sequence of Na⁺ >Li⁺ >K⁺, Rb⁺, Cs⁺, or tetramethylammonium, a sharp optimum at pH 7.2–7.3, activation by Ca²⁺, and loss of activity on aging which can be related to loss of endogenous K⁺. The reaction is nearly insensitive to either addition of exogenous Mg²⁺ or removal of membrane Mg²⁺ with EDTA. Each of these characteristics of passive swelling in acetate salts is duplicated in chloride media when tripropyltin is added to induce Cl^?/OH^? exchange. In contrast to nonenergized mitochondria, swelling of respiring mitochondria has been postulated to depend on electrophoretic uptake of cations in response to an interior negative membrane potential. Respiration-dependent swelling in acetate shows an indistinct cation selectivity sequence with Li⁺ and Na⁺ supporting higher rates of swelling at higher efficiency than K⁺, Rb⁺, and Cs⁺. The high rates of respiration-dependent swelling in Li⁺ and Na⁺ are inhibited by low levels of exogenous Mg²⁺ (K_i of 5–10 μm), but a significant swelling with almost no cation selectivity persists in the presences of 2 mm Mg²⁺. Removal of membrane Mg²⁺ by addition of EDTA strongly activates the rate of respiration-dependent swelling and converts a sigmoid dependency of swelling rate on Li⁺ concentration to a hyperbolic one with a Km of about 14 mm Li⁺. The cation selectivity and Mg²⁺ dependence of the reaction induced in chloride salts by tripropyltin are identical to these properties in acetate. Energy-dependent swelling in acetate shows optimum activity at pH 6.5 which appears related to the availability of free acetic acid, since the corresponding reaction induced in chloride shows a broad optimum at about pH 7.5. These studies support the concept that monovalent cations enter nonenergized mitochondria by electroneutral exchange with protons but penetrate respiring mitochondria by electrophoretic movement through one or more uniport pathways. They further suggest that both a Mg²⁺-sensitive uniport with high activity for Na⁺ and Li⁺ and a Mg²⁺-insensitive pathway with little cation discrimination are available in the membrane.     

The ribosome-associated complex antagonizes prion formation in yeast

The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI⁺] prion – an alternative conformer of Sup35 protein – and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Δzuo1 strains. Consistent with this finding, Δzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.