[Show abstract][Hide abstract] ABSTRACT: Yeast prions, based on self-seeded highly ordered fibrous aggregates (amyloids), serve as a model for human amyloid diseases.
Propagation of yeast prions depends on the balance between chaperones of the Hsp100 and Hsp70 families. The yeast prion [PSI+] can be eliminated by an excess of the chaperone Hsp104. This effect is reversed by an excess of the chaperone Hsp70-Ssa.
Here we show that the actions of Hsp104 and Ssa on [PSI+] are modulated by the small glutamine-rich tetratricopeptide cochaperone Sgt2. Sgt2 is conserved from yeast to humans, has
previously been implicated in the guided entry of tail-anchored proteins (GET) trafficking pathway, and is known to interact with Hsps, cytosolic Get proteins, and tail-anchored
proteins. We demonstrate that Sgt2 increases the ability of excess Ssa to counteract [PSI+] curing by excess Hsp104. Deletion of SGT2 also restores trafficking of a tail-anchored protein in cells with a disrupted GET pathway. One region of Sgt2 interacts
both with the prion domain of Sup35 and with tail-anchored proteins. Sgt2 levels are increased in response to the presence
of a prion when major Hsps are not induced. Our data implicate Sgt2 as an amyloid “sensor” and a regulator of chaperone targeting
to different types of aggregation-prone proteins.
[Show abstract][Hide abstract] ABSTRACT: Stigmatella aurantiaca displays a complex developmental life cycle in response to starvation conditions that results in the formation of tree-like fruiting bodies capable of producing spores. The phage Mx8, first isolated from the close relative Myxococcus xanthus, is unable to infect S. aurantiaca cells and integrate into the genome. However, plasmids containing Mx8 fragments encoding the integrase and attP are able to integrate at the attB locus in the S. aurantiaca genome by site-specific recombination. After recombination between attP and attB, the S. aurantiaca cells were incapable of building normal fruiting bodies but formed clumps and fungus-like structures characteristic of intermediate stages of development displayed by the wild type. We identified two tRNA genes, trnD and trnV, encoding tRNA(Asp) and tRNA(Val), respectively, composing an operon at the attB locus of S. aurantiaca. Integration of attP-containing plasmids resulted in the incorporation of the t(Mx8) terminator sequence, in addition to a short sequence of Mx8 DNA downstream of trnD. The integrant was unable to process the trnD transcript at the normal 3' processing site and displayed a lower level of expression of the trnVD operon. In addition, several developmentally regulated proteins were no longer produced in mutants following insertion at the attB locus. We hypothesize that the integration of the t(Mx8) terminator sequence results in reduced levels of mature tRNA(Asp) and tRNA(Val) and that altered protein production during development is thereby responsible for the observed phenotype. The trnVD locus thus defines a new developmental checkpoint for Stigmatella aurantiaca.
Journal of Bacteriology 04/2006; 188(5):1701-9. DOI:10.1128/JB.188.5.1701-1709.2006 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The yeast prion [PSI(+)] is a self-perpetuating aggregated isoform of the translation termination factor Sup35. [PSI ( + )] propagation is promoted by moderate levels and antagonized by high levels of the chaperone Hsp104. In agreement with the model postulating that excess Hsp104 acts on [PSI ( + )] by disaggregating prion polymers, we show that an increase in Sup35 levels, accompanied by an increase in size of prion aggregates, also partially protects [PSI(+)] from elimination by excess Hsp104. Despite retention of [PSI(+)], excess Hsp104 decreases toxicity of overproduced Sup35 in [PSI(+)] strains. A heritable variant of [PSI(+)], which has been isolated and is maintained only in the presence of increased levels of Hsp104, is characterized by an abnormally large aggregate size, and exhibits an altered response to overproduction of the Hsp70 chaperone Ssa1. These features resemble the previously described prion generated by a deletion derivative of Sup35, but are not associated with any sequence alteration and are controlled exclusively at the protein level. Our data provide a proof of the existence of conditionally stable prion variants maintained only at altered levels of Hsps, that could in principle be beneficial if the normal cellular function of a prion protein becomes detrimental to the cell in such conditions.
Current Genetics 02/2006; 49(1):21-9. DOI:10.1007/s00294-005-0035-0 · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: [PSI(+)] 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(+)]" and "anti-[PSI(+)]" effects, respectively. Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of [PSI(+)] and that pro-[PSI(+)] 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(+)]. Surprisingly, overproduction of either chaperone increases loss of [PSI(+)] 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.