Interspecies transmission of prions.

Mammalian prions are infectious agents of proteinaceous nature that cause several incurable neurodegenerative diseases. Interspecies transmission of prions is usually impeded or impossible. Barriers in prion transmission are caused by small interspecies differences in the primary structure of prion proteins. The barriers can also depend on the strain (variant) of a transmitted prion. Interspecies barriers were also shown for yeast prions, which define some heritable phenotypes. Yeast prions reproduce all the main traits of prion transmission barriers observed for mammals. This allowed to show that the barrier in prion transmission can be observed even upon copolymerization of two prionogenic proteins. Available data allow elucidation of the mechanisms that impede prion transmission or make it impossible.

In vitro propagation of the prion-like state of yeast Sup35 protein.

The yeast cytoplasmically inherited genetic determinant [PSI+] is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p). Here, cell-free conversion of Sup35p from [psi-] cells (Sup35ppsi-) to the prion-like [PSI+]-specific form (Sup35pPSI+) was observed. The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous [PSI+] propagation. Size fractionation of lysates of [PSI+] cells demonstrated that the converting activity was associated solely with Sup35pPSI+ aggregates, which agrees with the nucleation model for [PSI+] propagation. Sup35pPSI+ was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.

Chaperones that cure yeast artificial [PSI+] and their prion-specific effects.

The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.

Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation.

The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+] nonsense suppressor determinant. In a [PSI+] state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the [PSI+] phenotype. Sup35p is composed of the N-terminal domain (N) required for [PSI+] maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination. In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in [PSI+] cells. Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain. Similarly to Sup35p, in [PSI+] cells Sup45p was found in aggregates. The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding. The incorporation of Sup45p into the aggregates should inhibit its activity. The N domain of Sup35p, responsible for its aggregation in [PSI+] cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p. This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.

Wild type huntingtin toxicity in yeast: Implications for the role of amyloid cross-seeding in polyQ diseases.

Proteins with expanded polyglutamine (polyQ) regions are prone to form amyloids, which can cause diseases in humans and toxicity in yeast. Recently, we showed that in yeast non-toxic amyloids of Q-rich proteins can induce aggregation and toxicity of wild type huntingtin (Htt) with a short non-pathogenic polyglutamine tract. Similarly to mutant Htt with an elongated N-terminal polyQ sequence, toxicity of its wild type counterpart was mediated by induced aggregation of the essential Sup35 protein, which contains a Q-rich region. Notably, polymerization of Sup35 was not caused by the initial benign amyloids and, therefore, aggregates of wild type Htt acted as intermediaries in seeding Sup35 polymerization. This exemplifies a protein polymerization cascade which can generate a network of interdependent polymers. Here we discuss cross-seeded protein polymerization as a possible mechanism underlying known interrelations between different polyQ diseases. We hypothesize that similar mechanisms may enable proteins, which possess expanded Q-rich tracts but are not associated with diseases, to promote the development of polyQ diseases.

Further characterization of recessive suppression in yeast. Isolation of the low-temperature sensitive mutant of Saccharomyces cerevisiae defective in the assembly of 60 S ribosomal subunit.

It has been shown that recessive suppressor mutations in the yeast Saccharomyces cerevisiae may cause sensitivity towards low temperatures (very slow growth or lack of growth at 10 degrees C). One of the sup 1 low temperature sensitive (Lts-) mutants, 26-125A-P-2156, was studied in detail. After a prolonged period of incubation (70 h) under restrictive conditions the protein synthesis apparatus in the mutant cells was irreversibly damaged. In addition, Lts- cells incubated under restrictive conditions synthesize unequal amounts of ribosomal subunits, the level of 60 S subunit being reduced. It has been suggested that the recessive suppression is mediated by a mutation in the gene coding for 60 S subunit component, probably a ribosomal protein. The mutation leads simultaneously to a defect in the assembly of 60 S subunit and to low-temperature sensitive growth of the mutant.

Interaction of the yeast omnipotent suppressors SUP1(SUP45) and SUP2(SUP35) with non-mendelian factors.

The SUP1 and SUP2 genes code for protein factors intimately involved in the control of translational accuracy. The disrupted alleles of these genes confer a recessive lethal phenotype in both [psi+] and [psi-] genetic backgrounds, indicating an essential function for the corresponding proteins. In [psi+] diploids, heterozygous for the SUP1 null allele, several dominant phenotypes were evident with slow growth and inability to sporulate. These dominant phenotypes disappear after transformation with the multicopy plasmid carrying the wild-type allele of the SUP1 gene. Such dominant phenotypes were not observed for the SUP2 null allele. The incompatibility of multicopy plasmids carrying the SUP2 gene with guanidine hydrochloride-curable cytoplasmic factor(s) was also demonstrated. The possible mechanisms of interaction of the SUP1 and SUP2 genes with the [psi] determinant are discussed.

The SUP35 omnipotent suppressor gene is involved in the maintenance of the non-Mendelian determinant [psi+] in the yeast Saccharomyces cerevisiae.

The SUP35 gene of yeast Saccharomyces cerevisiae encodes a 76.5-kD ribosome-associated protein (Sup35p), the C-terminal part of which exhibits a high degree of similarity to EF-1 alpha elongation factor, while its N-terminal region is unique. Mutations in or overexpression of the SUP35 gene can generate an omnipotent suppressor effect. In the present study the SUP35 wild-type gene was replaced with deletion alleles generated in vitro that encode Sup35p lacking all or a part of the unique N-terminal region. These 5'-deletion alleles lead, in a haploid strain, simultaneously to an antisuppressor effect and to loss of the non-Mendelian determinant [psi+]. The antisuppressor effect is dominant while the elimination of the [psi+] determinant is a recessive trait. A set of the plasmid-borne deletion alleles of the SUP35 gene was tested for the ability to maintain [psi+]. It was shown that the first 114 amino acids of Sup35p are sufficient to maintain the [psi+] determinant. We propose that the Sup35p serves as a trans-acting factor required for the maintenance of [psi+].

Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae.

BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl - dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough. RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p. CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.

Nucleotide sequence of the SUP2 (SUP35) gene of Saccharomyces cerevisiae.

A nucleotide sequence of the yeast Saccharomyces cerevisiae omnipotent suppressor SUP2 (SUP35) gene is presented. The sequence contains a single open reading frame (ORF) of 2055 bp, which may encode a 76.5-kDa protein. A single transcript of 2.3 kb corresponding to a complete ORF is found. Analysis of codon bias suggests that the SUP2 gene is not highly expressed. The C-terminal part of the deduced amino acid sequence shows a high homology to yeast elongation factor EF-1 alpha, whereas the N-terminal part is unique for the SUP2 protein. The N terminus contains a number of short repeating elements and possesses an unusual amino acid composition. Analysis of the nucleotide and deduced amino acid sequences indicates that three additional proteins could possibly be expressed, two of which might be initiated on internal ATG codons and a third might be formed by alternative splicing. One of these proteins is supposed to be imported into mitochondria. Possible functions of the SUP2 gene product(s), especially its putative activity as a soluble factor controlling the fidelity of translation, are discussed.

Localization of possible functional domains in sup2 gene product of the yeast Saccharomyces cerevisiae.

Primary structures of yeast sup2 gene and polypeptide product coded by the gene are compared with the current nucleotide and amino acid sequence data base. The amino acid sequence of the sup2 product shows homology to elongation factors from different sources. Especially high homology is found in the regions, corresponding to conservative aminoacyl-tRNA- and GTP-binding domains, described in elongation factors and other proteins. The data obtained are discussed in relation to the functions of sup2 polypeptide product in protein synthesis.

Conservative system for dosage-dependent modulation of translational fidelity in eukaryotes.

Variations in dosage of some genes can alter the level of translational fidelity. The Saccharomyces cerevisiae genes that act as dosage-dependent suppressors and/or modulators of suppression, are the following: some tRNA genes (for example, tRNA(Gln)) inducing readthrough by mispairing; genes coding for either translational elongation factor or other proteins taking part in translation; and some genes of unknown function. We suggest that the SUP35 protein is a factor which may play a major role in balance-dependent regulation of translational fidelity. Homologues of this genes have been identified in other yeast genera (Pichia), green algae (Chlamydomonas) and various animals including man. No homologies have been found in the polychaeta (Nereis) or in insects (Drosophila). Rates of evolution differ for two separate parts of the genes; the N-terminal part, which is important for ambiguous translation in Saccharomyces, is markedly variable in the organisms tested. However, the C-terminal part which is required for yeast viability has a common origin but a separate evolution from that of the EF-Tu protein family.

Prions.

Prions were originally defined as infectious agents of protein nature, which caused neurodegenerative diseases in animals and humans. The prion concept implies that the infectious agent is a protein in special conformation that can be transmitted to the normal molecules of the same protein through protein-protein interactions. Until the 1990s, the prion phenomenon was associated with the single protein named PrP. Discovery of prions in lower eukaryotes, the yeast Saccharomyces cerevisiae and fungus Podospora anserina, suggests that prions have wider significance. Prions of lower eukaryotes are not related to diseases; their propagation caused by aggregation of prion-like proteins underlies the inheritance of phenotypic traits and most likely has adaptive significance. This review covers prions of mammals and lower eukaryotes, mechanisms of their appearance de novo and maintenance, structure of prion particles, and prospects for the treatment of prion diseases. Recent data concerning the search for new prion-like proteins is included. The paper focuses on the [PSI+] prion of S. cerevisiae, since at present it is the most investigated one. The biological significance of prions is discussed.

Inactivation of the Hansenula polymorpha PMR1 gene affects cell viability and functioning of the secretory pathway.

In yeast, functions of the endoplasmic reticulum (ER) depend on the Golgi apparatus Ca2+ pool, which is replenished by the medial-Golgi ion pump Pmr1p. Here, to dissect the role of the Golgi Ca2+ pool in protein folding and elimination of unfolded proteins in the ER, the manifestations of the pmr1 mutation in yeast Hansenula polymorpha were studied. The PMR1 gene was disrupted in a H. polymorpha diploid strain. Haploid segregants of this diploid bearing the disruption allele were viable, though they showed a severe growth defect on synthetic medium and rapidly died during storage at low temperature. Disruption of H. polymorpha PMR1 led to defects of the Golgi-hosted protein glycosylation and vacuolar protein sorting. This mutation increased the survival rate of H. polymorpha cells upon treatment with the proapoptotic drug amiodarone. Unlike Saccharomyces cerevisiae, the H. polymorpha pmr1 mutant was not hypersensitive to chemicals that induce the accumulation of unfolded proteins in the ER, indicating that the elimination of unfolded proteins from the ER was not essentially affected. At the same time, the pmr1 mutation improved the secretion of human urokinase and decreased its intracellular aggregation, indicating an influence of the mutation on the protein folding in the ER.

Prions: infectious proteins with genetic properties.

The data on prions--proteinaceous infectious agents--are briefly summarized. Prions cause several incurable neurodegenerative diseases in mammals, while in lower eukaryotes the prion properties of proteins may be responsible for the inheritance of some phenotypic traits. The novel experimental models for finding and studying proteins with prion properties based on the yeast Saccharomyces cerevisiae and the fungus Podospora anserina are described. The significance of the prion phenomenon for biology and medicine is discussed.

Ribosome-bound EF-1 alpha-like protein of yeast Saccharomyces cerevisiae.

The SUP2 (SUP35) omnipotent suppressor gene encodes the EF-1 alpha-like polypeptide, intimately involved in the control of translational ambiguity in the yeast Saccharomyces cerevisiae. The present study is devoted to the immunological characterization of the Sup2 protein. The SUP2 gene was fused to the Escherichia coli lacZ gene and a polyclonal antibody against the corresponding Sup2--beta-galactosidase hybrid protein was obtained. This antibody identified a 79-kDa protein that was absent in those cells where the SUP2 gene was disrupted, and an abundance of this protein was observed in cells overexpressing the SUP2 gene. The localization of this protein was studied in subcellular fractionation experiments. The SUP2 gene product proved to be uniformly distributed throughout ribosome-enriched samples, i.e. free polysomes, crude microsomes and rough endoplasmic reticulum. It was not found in the cytoplasm and smooth endoplasmic reticulum. The SUP2-encoded protein was fully ribosome associated and less abundant than the ribosomal protein L3. Also, in a sucrose gradient, Sup2 preferentially cosedimented with the 40S ribosomal subunit, but not with the 60S subunit. The functional significance of this association is discussed.

A disruption-replacement approach for the targeted integration of foreign genes in Hansenula polymorpha.

A system has been developed which allows the selection of integrative transformants with replacement of the Hansenula polymorpha methanol oxidase gene (MOX) with expression cassettes carrying heterologous gene under the control of the MOX promoter. The system is convenient for comparison of the expression levels of different constructs integrated into the same locus of the H. polymorpha genome. This system was used to compare the secretion levels of human urinary plasminogen activator, the secretion of which was directed by different signal sequences.

Isolation and characterization of the LEU2 gene of Hansenula polymorpha.

A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli. The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae. Further downstream, there is a partial reading frame with no obvious similarity to known proteins. The LEU2 gene of H. polymorpha cannot complement the leu2 mutation of S. cerevisiae.

Plasmid reorganization during integrative transformation in Hansenula polymorpha.

During studies of integrative transformation in Hansenula polymorpha, it was found that transformants with plasmids possessing the LEU2 gene of H. polymorpha were frequently unstable and lost plasmids while growing on non-selective medium. These transformants possessed reorganized plasmids capable of replication in H. polymorpha. Two such plasmids were isolated and characterized. It was shown that they contain additional DNA segments which were not present in the original plasmid used for transformation. Southern hybridization analysis carried out with labeled DNA probes derived from these segments showed that they consisted of H. polymorpha DNA. The hybridization patterns indicated that corresponding sequences were homologous to several chromosomal regions. These chromosomal DNA segments apparently carried H. polymorpha autonomous replicating sequences (HARS), since plasmids bearing them could transform H. polymorpha with high efficiency and were maintained in transformants in an autonomous state. Sequence analysis of one such captured chromosomal fragment revealed several eight- to ten-base AT-rich blocks similar to the presumed HARS sequence defined by Roggenkamp et al. (1986). Analogous reorganization was also observed with respect to integrative plasmids carrying the TRP3 and HIS3 genes of H. polymorpha and the ADE2 gene of Saccharomyces cerevisiae as selectable markers.