Hiroshi Kurahashi

[PHI+], a novel Sup35‐prion variant propagated with non‐Gln/Asn oligopeptide repeats in the absence of the chaperone protein Hsp104

Background: The [PSI+] element of the budding yeast is an aggregated form of the translation release factor Sup35 that is propagated and transmitted cytoplasmically in a manner analogous to that of mammalian prions. The N‐terminal of Sup35, necessary for [PSI+], contains oligopeptide repeats and multiple Gln/Asn residues.

Channel mutations in Hsp104 hexamer distinctively affect thermotolerance and prion-specific propagation

The yeast prion [PSI+] represents an aggregated state of the translation termination factor Sup35 resulting in the tendency of ribosomes to readthrough stop codons. In this study, we constructed an auxotrophic chromosomal marker, ura3‐197 (nonsense allele), applicable to selection for loss of [PSI+] to [psi–]. Unlike [psi–] yeast strains, [PSI+] yeast strains exhibit nonsense suppression of the ura3‐197 allele and are not viable in the presence of 5‐fluoroorotic acid (5‐FOA) that is converted to a toxic material by the readthrough product of Ura3. We selected 20 5‐FOA‐resistant, loss‐of‐[PSI+], mutants spontaneously or by transposon‐mediated mutagenesis from ura3‐197[PSI+] cells. All of the 20 [psi–] isolates were affected in Hsp104, a protein‐remodelling factor. Although most of them were disabled in a normal Hsp104 function for thermotolerance, three single mutants, L462R, P557L and D704N, remained thermotolerant. Importantly, L462R and D704N also eliminate other yeast prions [URE3] and [PIN+], while P557L does not, suggesting that Hsp104 harbours a unique activity to prion propagation independent of its function in thermotolerance. The mutations that are specific to prion propagation are clustered around the lateral channel of the Hsp104 hexamer, suggesting a crucial and specific role of this channel for prion propagation.

A regulatory role of the Rnq1 nonprion domain for prion propagation and polyglutamine aggregates.

ABSTRACT Prions are infectious, self-propagating protein conformations. Rnq1 is required for the yeast Saccharomyces cerevisiae prion [PIN+], which is necessary for the de novo induction of a second prion, [PSI+]. Here we isolated a [PSI+]-eliminating mutant, Rnq1Δ100, that deletes the nonprion domain of Rnq1. Rnq1Δ100 inhibits not only [PSI+] prion propagation but also [URE3] prion and huntingtins polyglutamine aggregate propagation in a [PIN+] background but not in a [pin−] background. Rnq1Δ100, however, does not eliminate [PIN+]. These findings are interpreted as showing a possible involvement of the Rnq1 prion in the maintenance of heterologous prions and polyQ aggregates. Rnq1 and Rnq1Δ100 form a sodium dodecyl sulfate-stable and Sis1 (an Hsp40 chaperone protein)-containing coaggregate in [PIN+] cells. Importantly, Rnq1Δ100 is highly QN-rich and prone to self-aggregate or coaggregate with Rnq1 when coexpressed in [pin−] cells. However, the [pin−] Rnq1-Rnq1Δ100 coaggregate does not represent a prion-like aggregate. These findings suggest that [PIN+] Rnq1-Rnq1Δ100 aggregates interact with other transmissible and nontransmissible amyloids to destabilize them and that the nonprion domain of Rnq1 plays a crucial role in self-regulation of the highly reactive QN-rich prion domain of Rnq1.

Tropomyosin is required for the cell fusion process during conjugation in fission yeast

Background:  Tropomyosin is an actin‐binding protein, which is thought to stabilize actin filaments and influence many aspects of F‐actin. In fission yeast, the cdc8 gene encodes tropomyosin, and the gene product Cdc8p is known to be essential for the formation of the F‐actin contractile ring and hence for cytokinesis in the mitotic cell cycle.

A Systematic Evaluation of the Function of the Protein-Remodeling Factor Hsp104 in [PSI+] Prion Propagation in S. cerevisiae by Comprehensive Chromosomal Mutations

The yeast prion [PSI+] represents an aggregated state of the translational release factor Sup35 (eRF3) and deprives termination complexes of functional Sup35, resulting in nonsense codon suppression. Protein-remodeling factor Hsp104 is involved in thermotolerance and [PSI+] propagation, however the structure-and-function relationship of Hsp104 for [PSI+] remains unclear. In this study, we engineered 58 chromosomal hsp104 mutants that affect residues considered structurally or functionally relevant to Hsp104 remodeling activity, yet most remain to be examined for their significance to [PSI+] in the same genetic background. Many of these hsp104 mutants were affected both in thermotolerance and [PSI+] propagation. However, nine mutants were impaired exclusively for [PSI+], while two mutants were impaired exclusively for thermotolerance. Mutations exclusively affecting [PSI+] are clustered around the lateral channel of the Hsp104 hexamer. These findings suggest that Hsp104 possesses shared as well as distinct remodeling activities for stress-induced protein aggregates and [PSI+] prion aggregates and that the lateral channel plays a role specific to [PSI+] prion propagation.

[PSI+] aggregate enlargement in rnq1 nonprion domain mutants, leading to a loss of prion in yeast

[PIN+] is the prion form of the Rnq1 protein of unknown function in Saccharomyces cerevisiae. A glutamine/asparagine (Q/N)‐rich C‐terminal domain is necessary for the propagation of [PIN+], whereas the N‐terminal region is non‐Q/N‐rich and considered the nonprion domain. Here, we isolated numerous single‐amino‐acid mutations in Rnq1, phenotypically similar to Rnq1Δ100, which inhibit [PSI+] propagation in the [PIN+] state, but not in the [pin−] state, when overproduced. The dynamics of the prion aggregates was analyzed by semi‐denaturing detergent‐agarose gel electrophoresis and fluorescence correlation spectroscopy. The results indicated that [PSI+] aggregates were enlarged in mother cells and, instead, not apparently transmitted into daughter cells. Under these conditions, the activity of Hsp104, a known prion disaggregase, was not affected when monitored for the thermotolerance of the rnq1 mutants. These [PSI+]‐inhibitory rnq1 mutations did not affect [PIN+] propagation itself when over‐expressed from a strong promoter, but instead destabilized [PIN+] when expressed from the weak authentic RNQ1 promoter. The majority of these mutated residues are mapped to the surface, and on one side, of contiguous α‐helices of the nonprion domain of Rnq1, suggesting its involvement in interactions with a prion or a factor necessary for prion development.

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, over-expression 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.

A G-protein γ subunit mimic is a general antagonist of prion propagation in Saccharomyces cerevisiae

The Gpg1 protein is a Gγ subunit mimic implicated in the G-protein glucose-signaling pathway in Saccharomyces cerevisiae, and its function is largely unknown. Here we report that Gpg1 blocks the maintenance of [PSI+], an aggregated prion form of the translation termination factor Sup35. Although the GPG1 gene is normally not expressed, over-expression of GPG1 inhibits propagation of not only [PSI+] but also [PIN+], [URE3] prions, and the toxic polyglutamine aggregate in S. cerevisiae. Over-expression of Gpg1 does not affect expression and activity of Hsp104, a protein-remodeling factor required for prion propagation, showing that Gpg1 does not target Hsp104 directly. Nevertheless, prion elimination by Gpg1 is weakened by over-expression of Hsp104. Importantly, Gpg1 protein is prone to self-aggregate and transiently colocalized with Sup35NM-prion aggregates when expressed in [PSI+] cells. Genetic selection and characterization of loss-of-activity gpg1 mutations revealed that multiple mutations on the hydrophobic one-side surface of predicted α-helices of the Gpg1 protein hampered the activity. Prion elimination by Gpg1 is unaffected in the gpa2Δ and gpb1Δ strains lacking the supposed physiological G-protein partners of Gpg1. These findings suggest a general inhibitory interaction of the Gpg1 protein with other transmissible and nontransmissible amyloids, resulting in prion elimination. Assuming the ability of Gpg1 to form G-protein heterotrimeric complexes, Gpg1 is likely to play a versatile function of reversing the prion state and modulating the G-protein signaling pathway.

A bipolar functionality of Q/N-rich proteins: Lsm4 amyloid causes clearance of yeast prions

Prions are epigenetic modifiers that cause partially loss‐of‐function phenotypes of the proteins in Saccharomyces cerevisiae. The molecular chaperone network that supports prion propagation in the cell has seen a great progress in the last decade. However, the cellular machinery to activate or deactivate the prion states remains an enigma, largely due to insufficient knowledge of prion‐regulating factors. Here, we report that overexpression of a [PSI+]‐inducible Q/N‐rich protein, Lsm4, eliminates the three major prions [PSI+], [URE3], and [RNQ+]. Subcloning analysis revealed that the Q/N‐rich region of Lsm4 is responsible for the prion loss. Lsm4 formed an amyloid in vivo, which seemed to play a crucial role in the prion elimination. Fluorescence correlation spectroscopy analysis revealed that in the course of the Lsm4‐driven [PSI+] elimination, the [PSI+] aggregates undergo a size increase, which ultimately results in the formation of conspicuous foci in otherwise [psi−]‐like mother cells. We also found that the antiprion activity is a general property of [PSI+]‐inducible factors. These data provoked a novel “unified” model that explains both prion induction and elimination by a single scheme.

A bipolar personality of yeast prion proteins.

Prions are infectious, self-propagating protein conformations. [PSI+], [RNQ+] and [URE3] are well characterized prions in Saccharomyces cerevisiae and represent the aggregated states of the translation termination factor Sup35, a functionally unknown protein Rnq1, and a regulator of nitrogen metabolism Ure2, respectively. Overproduction of Sup35 induces the de novo appearance of the [PSI+] prion in [RNQ+] or [URE3] strain, but not in non-prion strain. However, [RNQ+] and [URE3] prions themselves, as well as overexpression of a mutant Rnq1 protein, Rnq1Δ100, and Lsm4, hamper the maintenance of [PSI+]. These findings point to a bipolar activity of [RNQ+], [URE3], Rnq1Δ100, and Lsm4, and probably other yeast prion proteins as well, for the fate of [PSI+] prion. Possible mechanisms underlying the apparent bipolar activity of yeast prions will be discussed.