The extrachromosomal control of nonsense suppression in yeast: an analysis of the elimination of [psi+] in the presence of a nuclear gene PNM.

Summary When a [psi ^-] strain of yeast mutates to [psi ⁺], the efficiency of suppression by certain ochre suppressors is increased. The [psi ⁺] phenotype is inherited extrachromosomally. There is a nuclear gene, PNM, which, when mutant, causes loss of the [psi ⁺] phenotype. PNM ^- is dominant to PNM ⁺ and a heterozygous diploid gradually loses the ability over successive generations, to produce PNM ⁺ [psi ⁺] spores. This paper describes the kinetics of this elimination and the data obtained are discussed in relation to two models of the molecular nature of the [psi] genetic determinant—one considering the [psi] determinant as an autonomous nucleic acid, the other treating the possibility that the [psi] nucleic acid is that which codes for rRNA in the nuclear genome.     

Relationship of the [psi] factor with other plasmids of Saccharomyces cerevisiae

The [psi] factor is an extrachromosomally inherited genetic determinant of the yeast Saccharomyces cerevisiae for which no autonomous physical determinant has yet been identified. Using both physical and genetical techniques we demonstrate that the [psi] determinant does not reside on other previously described yeast extrachromosomally inherited determinants; namely mitochondrial DNA, L double-stranded RNA, M double-stranded RNA, and [2 μm] DNA. Stable [psi⁺] strains, lacking one or other of these determinants were constructed. It is concluded that the [psi] factor may represent a new yeast plasmid.     

Ribosomal proteins of yeast strains carrying mutations which affect the efficiency of nonsense suppression

Summary We have examined the ribosomal proteins of strains of Saccharomyces cerevisiae which differ in the efficiency with which ochre nonsense mutations are suppressed. The strains in which ochre suppression is poor were [psi]^- or carried antisuppressor mutations; those in which suppression was highly efficient were [psi]⁺ or carried allosuppressor mutations. The ribosomal proteins of these strains, as judged by two-dimensional polyacrylamide gel electrophoresis, were indistinguishable from those of wild-type.     

Ultraviolet Mutagenesis Studies of [psi], a Cytoplasmic Determinant of SACCHAROMYCES CEREVISIAE

UV mutagenesis was used to probe the molecular nature of [psi], a non-mitochondrial cytoplasmic determinant of Saccharomyces cerevisiae involved in the control of nonsense suppression. The UV-induced mutation from [psi⁺] to [psi^-] showed characteristics of forward nuclear gene mutation in terms of frequency, induction kinetics, occurrence of whole and sectored mutant clones and the effect of the stage in the growth cycle on mutation frequency. The involvement of pyrimidine dimers in the premutational lesion giving the [psi^-] mutation was demonstrated by photoreactivation. UV-induced damage to the [psi] genetic determinant was shown to be repaired by nuclear-coded repair enzymes that are responsible for the repair of nuclear DNA damage. UV-induced damage to mitochondrial DNA appeared to be, at least partly, under the control of different repair processes. The evidence obtained suggests that the [psi] determinant is DNA.     

The effects of paromomycin on the fidelity of translation in a yeast cell-free system

The effects of the aminoglycoside antibiotic paromomycin on the fidelity of translation of the synthetic template poly(U), and two natural mRNAs (rabbit globin mRNA and Brome Mosaic virus RNA), were examined in an mRNA-dependent cell-free system from the yeast Saccharomyces cerevisiae. At antibiotic concentrations that did not inhibit translation (100 μM) optimal mistranslation of all three templates was observed, with the effects declining at higher antibiotic concentrations. Synthesis of the opal termination read-through protein of rabbit β-globin mRNA was induced by paromomycin, but only in lysates prepared from a [psi⁺] strain of yeast. The antibiotic did not induce detectable levels of either ochre or amber read-through, but did induce general misreading of Brome Mosaic virus RNA to the same degree in both [psi⁺] and [psi^?] lysates. This misreading was enhanced by addition of the polyamine spermidine.     

Prion-Dependent Lethality of sup45 Mutants in Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae translation termination factors eRF1 (Sup45) and eRF3 (Sup35) are encoded by the essential genes SUP45 and SUP35 respectively. Heritable aggregation of Sup35 results in formation of the yeast prion [PSI⁺]. It is known that combination of [PSI⁺] with some mutant alleles of the SUP35 or SUP45 genes in one and the same haploid yeast cell causes synthetic lethality. In this study, we perform detailed analysis of synthetic lethality between various sup45 nonsense and missense mutations on one hand, and different variants of [PSI⁺] on the other hand. Synthetic lethality with sup45 mutations was detected for [PSI⁺] variants of different stringencies. Moreover, we demonstrate for the first time that in some combinations, synthetic lethality is dominant and occurs at the postzygotic stage after only a few cell divisions. The tRNA suppressor SUQ5 counteracts the prion-dependent lethality of the nonsense alleles but not of the missense alleles of SUP45, indicating that the lethal effect is due to the depletion of Sup45. Synthetic lethality is also suppressed in the presence of the C-proximal fragment of Sup35 (Sup35C) that lacks the prion domain and cannot be included into the prion aggregates. Remarkably, the production of Sup35C in a sup45 mutant strain is also accompanied by an increase in the Sup45 levels, suggesting that translationally active Sup35 up-regulates Sup45 or protects it from degradation.Key Words: Sup45, Sup35, eRF1, eRF3, amyloid, [PSI⁺], translation termination, Saccharomyces cerevisiae     

Serine substitutions caused by an ochre suppressor in yeast.

The suppressor SUQ5 in yeast can cause the production of approximately 10 to 20% of the normal amount of iso-l-cytochrome c when coupled to the ochre (UAA) mutants cyc1–2 and cyc1–72. The iso-l-cytochromes c contain residues of serine at positions that correspond to the sites of the ochre codons. SUQ5 is efficient only in strains having the non-Mendelian factor ψ⁺, although the low amount of suppressed iso-l-cytochrome c from a ψ⁻SUQ5 cyc1–72 strain was also shown to contain serine at the ochre site. Thus SUQ5 differs from the eight other characterized suppressors of UAA in yeast, which were previously shown to insert residues of tyrosine at ochre sites (Gilmore et al., 1971) and which are only effective in strains haying the non-Mendelian factor ψ⁻, since they generally cause inviability in the ψ⁺ state. Like the tyrosine-inserting suppressors, SUQ5 can also act on another ochre allele cyc1–9, but with a very low efficiency of approximately 0.4%, while it does not appear to act at all on amber (UAG) mutants. SUQ5 was found to be 6.4 cM (centiMorgans) from tyr7 on chromosome XVI. It is suggested that the gene product of SUQ5 is serine tRNA.     

Over‐expression of the molecular chaperone Hsp104 in Saccharomyces cerevisiae results in the malpartition of [PSI+] propagons

The ability of a yeast cell to propagate [PSI⁺], the prion form of the Sup35 protein, is dependent on the molecular chaperone Hsp104. Inhibition of Hsp104 function in yeast cells leads to a failure to generate new propagons, the molecular entities necessary for [PSI⁺] propagation in dividing cells and they get diluted out as cells multiply. Over‐expression of Hsp104 also leads to [PSI⁺] prion loss and this has been assumed to arise from the complete disaggregation of the Sup35 prion polymers. However, in conditions of Hsp104 over‐expression in [PSI⁺] cells we find no release of monomers from Sup35 polymers, no monomerization of aggregated Sup35 which is not accounted for by the proportion of prion‐free [psi^‐] cells present, no change in the molecular weight of Sup35‐containing SDS‐resistant polymers and no significant decrease in average propagon numbers in the population as a whole. Furthermore, they show that over‐expression of Hsp104 does not interfere with the incorporation of newly synthesised Sup35 into polymers, nor with the multiplication of propagons following their depletion in numbers while growing in the presence of guanidine hydrochloride. Rather, they present evidence that over‐expression of Hsp104 causes malpartition of [PSI⁺] propagons between mother and daughter cells in a sub‐population of cells during cell division thereby generating prion‐free [psi^?] cells.     

Interaction of Human Laminin Receptor with Sup35, the [PSI +] Prion-Forming Protein from S. cerevisiae: A Yeast Model for Studies of LamR Interactions with Amyloidogenic Proteins

The laminin receptor (LamR) is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP^C and PrP^Sc. Indeed, LamR is a receptor for PrP^C. Whether LamR interacts with PrP^Sc exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP^Sc, is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP) were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI ⁺] and [psi ⁻]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI ⁺] strains. The presence of [PSI ⁺] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI ⁺] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI ⁺] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases.     

Requirements of Hsp104p activity and Sis1p binding for propagation of the [RNQ+] prion

The formation and maintenance of prions in the yeast Saccharomyces cerevisiae is highly regulated by the cellular chaperone machinery. The most important player in this regulation is Hsp104p, which is required for the maintenance of all known prions. The requirements for other chaperones, such as members of the Hsp40 or Hsp70 families, vary with each individual prion. [RNQ⁺] cells do not have a phenotype that is amenable to genetic screens to identify cellular factors important in prion propagation. Therefore, we used a chimeric construct that reports the [RNQ⁺] status of cells to perform a screen for mutants that are unable to maintain [RNQ⁺]. We found eight separate mutations in Hsp104p that caused [RNQ⁺] cells to become [rnq⁻]. These mutations also caused the loss of the [PSI⁺] prion. The expression of one of these mutants, Hsp104p-E190K, showed differential loss of the [RNQ⁺] and [PSI⁺] prions in the presence of wild type Hsp104p. Hsp104p-E190K inefficiently propagated [RNQ⁺] and was unable to maintain [PSI⁺]. The mutant was unable to act on other in vivo substrates, as strains carrying it were not thermotolerant. Purified recombinant Hsp104p-E190K showed a reduced level of ATP hydrolysis as compared to wild type protein. This is likely the cause of both prion loss and lack of in vivo function. Furthermore, it suggests that [RNQ⁺] requires less Hsp104p activity to maintain transmissible protein aggregates than Sup35p. Additionally, we show that the L94A mutation in Rnq1p, which reduces its interaction with Sis1p, prevents Rnq1p from maintaining a prion and inducing [PSI⁺].Key words: [RNQ⁺], [PSI⁺], Hsp104p, Sis1p, mutagenesis     

Localization of prion-destabilizing mutations in the N-terminal non-prion domain of Rnq1 in Saccharomyces cerevisiae

[PIN⁺] is the prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [PSI⁺]. The function of Rnq1, however, is little understood. The limited availability of defective rnq1 alleles impedes the study of its structure-function relationship by genetic analysis. In this study, we isolated rnq1 mutants that are defective in the stable maintenance of the [PIN⁺] prion. Since there is no rnq1 phenotype available that is applicable to a direct selection or screening for loss-of-function rnq1 mutants, we took advantage of a prion inhibitory agent, Rnq1Δ100, to develop a color-based genetic screen. Rnq1Δ100 eliminates the [PSI⁺] prion in the [PIN⁺] state but not in the [pin⁻] state. This allows us to find loss-of-[PIN⁺] rnq1 mutants as white [PSI⁺] colonies. Nine rnq1 mutants with single-amino-acid substitutions were defined. These mutations impaired the stable maintenance of [PIN⁺] and, as a consequence, were also partially defective in the de novo induction of [PSI⁺]. Interestingly, eight of the nine alleles were mapped to the N-terminal region of Rnq1, which is known as the non-prion domain preceding the asparagine and glutamine rich prion domain of Rnq1. Notably, overexpression of these rnq1 mutant proteins restored [PIN⁺] prion activity, suggesting that each of the rnq1 mutants was not completely inactive. These findings indicate that the N-terminal non-prion domain of Rnq1 harbors a potent activity to regulate the maintenance of the [PIN⁺] prion.Key words: Rnq1, [PIN⁺], Sup35, [PSI⁺], yeast prion     

Relationship between Intracellular Na+ Concentration and Reduced Na+ Affinity in Na+,K+-ATPase Mutants Causing Neurological Disease

The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na⁺,K⁺-ATPase α₂ and α₃ isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na⁺,K⁺-ATPase mutations, extending the C terminus by either 28 residues (“+28” mutation) or an extra tyrosine (“+Y”), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na⁺ and K⁺ concentrations ([Na⁺]_i and [K⁺]_i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na⁺ affinity without disturbance of K⁺ binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α₂ despite a high expression level. A significant rise of [Na⁺]_i and reduction of [K⁺]_i was detected in cells expressing mutants with reduced Na⁺ affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na⁺ affinity were found to reduce [Na⁺]_i. It is concluded that the Na⁺ affinity of the Na⁺,K⁺-ATPase is an important determinant of [Na⁺]_i.     

Influence of the processing of MucA protein on the reversion of the frameshift hisD3052 and the base substitution hisG46 mutations by chemical mutagens in Salmonella typhimurium

The correlation between the proficiency at promoting mutagenesis of MucA/B proteins and MucA processing has been considered to be very high (Hauser et al., 1992) on the basis of the results of UV mutagenicity (Shiba et al., 1990). Here we show that this correlation is only partial. We have assayed the mutagenicity of benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1) in Salmonella typhimurium tester strains containing plasmids which encode MucA proteins with an altered cleavage site. Reversion of the frameshift hisD3052 mutation by B[a]P or AFB1 was observed in the presence of non-cleavable MucA protein although at a lower level than that found in cells containing wild-type MucA protein. Reversion of the base substitution hisG46 mutation by AFB1 requires a significant processing of MucA, while lower levels of this processing would be enough for the hisG46 reversion by B[a]P. These results suggest that the specificity of mutations induced by mutagens forming DNA adducts is influenced by the activity of MucA protein. They also show the relevance of mutagenicity assays in the mechanistic studies of mutagenesis.     

Metabolic Inhibition Strongly Inhibits Na-Dependent Mg Efflux in Rat Ventricular Myocytes

We measured intracellular Mg²⁺ concentration ([Mg²⁺]_i) in rat ventricular myocytes using the fluorescent indicator furaptra (25°C). In normally energized cells loaded with Mg²⁺, the introduction of extracellular Na⁺ induced a rapid decrease in [Mg²⁺]_i: the initial rate of decrease in [Mg²⁺]_i (initial Δ[Mg²⁺]_i/Δt) is thought to represent the rate of Na⁺-dependent Mg²⁺ efflux (putative Na⁺/Mg²⁺ exchange). To determine whether Mg²⁺ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na⁺ gradient, we estimated the initial Δ[Mg²⁺]_i/Δt after metabolic inhibition. In the absence of extracellular Na⁺ and Ca²⁺, treatment of the cells with 1 μM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg²⁺]_i from ∼0.9 mM to ∼2.5 mM in a period of 5-8 min (probably because of breakdown of MgATP and release of Mg²⁺) and cell shortening to ∼50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg²⁺]_i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for ≥90 min. The initial Δ[Mg²⁺]_i/Δt was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59-85 min), a significant decrease in the initial Δ[Mg²⁺]_i/Δt (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na⁺ concentration ([Na⁺]_i) estimated with a Na⁺ indicator sodium-binding benzofuran isophthalate was, on average, 5.0-10.5 mM during the time required for the initial Δ[Mg²⁺]_i/Δt measurements, which is well below the [Na⁺]_i level for half inhibition of the Mg²⁺ efflux (∼40 mM). Normalization of intracellular pH using 10 μM nigericin, a H⁺ ionophore, did not reverse the inhibition of the Mg²⁺ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (∼0.4 mM estimated indirectly in the this study), rather than elevation of [Na⁺]_i or intracellular acidosis, inhibits the Mg²⁺ efflux, suggesting the absolute necessity of ATP for the Na⁺/Mg²⁺ exchange.     

Ssa1 Overexpression and [PIN] Variants Cure [PSI] by Dilution of Aggregates

Several cellular chaperones have been shown to affect the propagation of the yeast prions [PSI⁺], [PIN⁺] and [URE3]. Ssa1 and Ssa2 are Hsp70 family chaperones that generally cause pro-[PSI⁺] effects, since dominant-negative mutants of Ssa1 or Ssa2 cure [PSI⁺], and overexpression of Ssa1 enhances de novo [PSI⁺] appearance and prevents curing by excess Hsp104. In contrast, Ssa1 was shown to have anti-[URE3] effects, since overexpression of Ssa1 cures [URE3]. Here we show that excess Ssa1 or Ssa2 can also cure [PSI⁺]. This curing is enhanced in the presence of [PIN⁺]. During curing, Sup35-GFP fluorescent aggregates get bigger and fewer in number, which leads to their being diluted out during cell division, a phenotype that was also observed during the curing of [PSI⁺] by certain variants of [PIN⁺]. The sizes of the detergent-resistant [PSI⁺] prion oligomers increase during [PSI⁺] curing by excess Ssa1. Excess Ssa1 likewise leads to an increase in oligomer sizes of low, medium and very high [PIN⁺] variants. While these phenotypes are also caused by inhibition of Hsp104 or Sis1, the overexpression of Ssa1 did not cause any change in Hsp104 or Sis1 levels.     

Comparison of crude lysate pellets from isogenic strains of yeast with different prion composition: Identification of prion-associated proteins

A new approach involving the comparative analysis of proteins of crude cell lysate pellets from isogenic strains of Saccharomyces cerevisiae distinguished by their prion composition permitted us to identify a large group of prion-associated proteins in yeast cells. 35 proteins whose aggregation state depends on prion content have been identified by 2D-electrophoresis followed by the MALDI analysis of a recipient [psi ⁻] strain and of [PSI ⁺] cytoductant. Approximately half of these proteins belong to functional groups of chaperones and enzymes involved in glucose metabolism. Other proteins are involved in translation, stress response and protein degradation. The data obtained are compared with the results of other groups who used different approaches to detect proteins involved in prion aggregates.     

Search for genes influencing the maintenance of the [<Emphasis Type="Italic">ISP</Emphasis><Superscript>+</Superscript>] prion-like antisuppressor determinant in yeast with the use of an insertion gene library

The prion-like determinant [ISP ⁺] manifests itself as an antisuppressor of certain sup35 mutations. To establish that [ISP ⁺] is indeed a new yeast prion, it is necessary to identify the gene that codes for the protein whose prion form is [ISP ⁺]. Analysis of the transformants obtained by transformation of an [ISP ⁺] strain with an insertion gene library revealed three genes controlling the [ISP ⁺] maintenance: UPF1, UPF2, and SFP1. SFP1 codes for a potentially prionogenic protein, which is enriched in Asn and Gln residues, and is thereby the most likely candidate for the [ISP ⁺] structural gene. UPF1 and UPF2 code for components of nonsense-mediated mRNA decay. The [ISP ⁺] elimination caused by UPF1 and UPF2 inactivation was reversible, and Upf1p and Upf2p were not functionally related to phosphatase Ppz1p, which influences the [ISP ⁺] manifestation. Possible mechanisms sustaining the influence of UPF1 and UPF2 on [ISP ⁺] maintenance are discussed.