Academic journal article Genetics

Hsp70 Chaperones as Modulators of Prion Life Cycle: Novel Effects of SSA and Ssb on the Saccharomyces Cerevisiae Prion [PSI^sup +^]

Academic journal article Genetics

Hsp70 Chaperones as Modulators of Prion Life Cycle: Novel Effects of SSA and Ssb on the Saccharomyces Cerevisiae Prion [PSI^sup +^]

Article excerpt

ABSTRACT

[PSI^sup +^] is a prion isoform of the yeast release factor Sup35. In some assays, the cytosolic chaperones Ssa1 and Ssb1/2 of the Hsp70 family were previously shown to exhibit "pro-[PSI^sup +^]" and "anti-[PSI^sup +^]" effects, respectively. Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of [PSI^sup +^] and that pro-[PSI^sup +^] effects of Ssa1 are shared by all other Ssa proteins. Experiments with chimeric constructs show that the peptide-binding domain is a major determinant of differences in the effects of Ssa and Ssb proteins on [PSI^sup +^]. Surprisingly, overproduction of either chaperone increases loss of [PSI^sup +^] when Sup35 is simultaneously overproduced. Excess Ssa increases both the average size of prion polymers and the proportion of monomeric Sup35 protein. Both in vivo and in vitro experiments uncover direct physical interactions between Sup35 and Hsp70 proteins. The proposed model postulates that Ssa stimulates prion formation and polymer growth by stabilizing misfolded proteins, which serve as substrates for prion conversion. In the case of very large prion aggregates, further increase in size may lead to the loss of prion activity. In contrast, Ssb either stimulates refolding into nonprion conformation or targets misfolded proteins for degradation, in this way counteracting prion formation and propagation.

PRIONS are infectious or heritable agents transmitted at the protein level. Prions cause neurodegenerative diseases, such as "mad cow" and Creutzfeldt-Jacob diseases, in mammals and humans (for review, see PRUSINER et al. 1998). Prion and nonprion (cellular) isoforms of one and the same protein can have identical amino acid sequences but differ from each other in conformation and ability to form aggregated multimolecular structures. Mammalian prions are usually aggregationprone proteins, capable of generating fiber-like polymers of ordered structure, called amyloids. This resembles noninfectious amyloidoses and inclusion body disorders, such as Alzheimer's or Huntington's diseases. The model of "nucleated polymerization" (for review, see LANSBURY and CAUGHEY 1995) proposes that prion reproduction occurs via shearing of the large aggregates into oligomeric "seeds" that grow by immobilizing the newly synthesized protein molecules. An alternative model of "template assistance" (for review, see HARRISON et al 1997) views prion seeds as misfolded monomers that stimulate misfolding of the newly synthesized or partially unfolded polypeptides of the same amino acid sequence.

Several proteins of different structures and functions can form prions in yeast, as concluded first by WICKNER (1994) on the basis of genetic criteria and confirmed by subsequent genetic and biochemical experiments (for reviews, see CHERNOFF 2001, 2004). Known yeast amyloidogenic prion proteins include [PSI^sup +^] (Cox 1965), a prion isoform of the translation termination factor Sup35 (eRF3); [URE3] (LACROUTE 1971), a prion isoform of Ure2, the regulatory protein in nitrogen metabolism; and [PIN^sup +^] (DERKATCH et al 1997), or [RNQ^sup +^] (SONDHEIMER and LINDQUIST 2000; DERKATCH et al 2001), a prion form of Rnql, a protein of an unknown function. Yeast prions provide a molecular mechanism for protein-based inheritance by controlling phenotypic traits inherited in a non-Mendelian fashion (for reviews, see CHERNOFF 2001, 2004).

As prion formation and propagation is apparently modulated at the level of three-dimensional and/or quarternary structure of a protein, chaperone proteins play an important role in these processes. The yeast prion [PSI^sup +^] is eliminated by overproduction or inactivation of the chaperone Hspl04 (CHERNOFF et al. 1995). As Hsp104 is involved in disaggregation of heat-damaged proteins (PARSELL et al. 1994; GLOVER and LINDQUIST 1998), it has been hypothesized that moderate levels of Hsp104 generate new prion seeds via shearing of the preexisting prion aggregates, while high levels of Hsp104 eliminate prion aggregates by converting them into nonprion monomers (PAUSHKIN et al. …

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