RESUMO
The yeast prions represent a very attractive and tractable model for investigating the prion world. The more extensively studied yeast prion [PSI] leads to a propagation model that links auto-aggregation in amyloid formation and inactivation of the cellular function of the yeast 'prion protein' Sup35p. The other prion model, [URE3], appears to be similar in some genetic and biochemical properties. The characterisation of both Sup35p and Ure2p, the two 'prion proteins', mainly focusing on their aggregation properties, support this model. However, some important differences still exist that should be examined carefully. In particular, we have shown that Ure2p aggregation in vivo (monitored by fluorescence of Ure2-GFP fusion) does not necessarily give rise to a [URE3] phenotype. Comparisons of these two systems as well as more recent experiments are discussed in this review.
Assuntos
Proteínas Fúngicas/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Animais , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glutationa Peroxidase , Proteínas de Fluorescência Verde , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Fatores de Terminação de Peptídeos , Fenótipo , Príons/química , Príons/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The non-Mendelian element [URE3] of yeast is considered to be a prion form of the Ure2 protein. The [URE3] phenotype occurs at a frequency of 10(-5) in haploid yeast strains, is reversible, and its frequency is increased by overexpressing the URE2 gene. We created a new mutant of the Ure2 protein, called H2p, which results in a 1000-fold increase in the rate of [URE3] occurrence. To date, only the overexpression of various C-terminal truncated mutants of Ure2p gives rise to a comparable level. The h2 allele is, thus, the first characterized URE2 allele that induces prion formation when expressed at a low level. By shuffling mutated and wild-type domains of URE2, we also created the first mutant Ure2 protein that is functional and induces prion formation. We demonstrate that the domains of URE2 function synergistically in cis to induce [URE3] formation, which highlights the importance of intramolecular interactions in Ure2p folding. Additionally, we show using a green fluorescent protein (GFP) fusion protein that the h2 allele exhibits numerous filiform structures that are not generated by the wild-type protein.
Assuntos
Alelos , Proteínas Fúngicas/genética , Príons/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Bases , Primers do DNA , Glutationa Peroxidase , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Mutagênese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.
Assuntos
Proteínas Fúngicas/química , Príons , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Sequência de Bases , Biopolímeros , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Proteínas Fúngicas/genética , Glutationa Peroxidase , Microscopia Eletrônica , Peso Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , SolubilidadeRESUMO
In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.
