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Reed B Wickner

Publications and source records attributed to Reed B Wickner.

At least 19 recordsLinked to original sources
PubMed2006

Amyloid of the prion domain of Sup35p has an in-register parallel beta-sheet structure.

The [PSI(+)] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the structure of amyloid fibrils formed in vitro from purified recombinant Sup35(1-253), consisting of the glutamine- and asparagine-rich N-terminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole-dipole couplings among (13)C nuclei in a series of Sup35NM fibril samples, (13)C-labeled at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular (13)C-(13)C distances of approximately 0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of approximately 0.5 nm. These results indicate that an in-register parallel beta-sheet structure underlies the [PSI(+)] prion phenomenon.

Amino Acid Sequence↗
PubMed2006

Nitrogen source and the retrograde signalling pathway affect detection, not generation, of the [URE3] prion.

[URE3] is an infectious (prion) inactive amyloid form of Ure2p, a regulator of nitrogen catabolism. [URE3] clones are selected on NH(4) (+), using their derepressed expression of DAL5 to allow uptake of ureidosuccinate (USA). We previously reported that mks1Delta prevents generation of [URE3] and others reported that glutamate in the medium or the elevated glutamate in mks1Delta strains blocks [URE3] generation. We show here that elevated glutamate does not block [URE3] generation, but that neither does mks1Delta. Rather, a post-transcriptional effect on DAL5 of mks1Delta through the retrograde regulation pathway prevents detection of [URE3] prion-containing colonies. Moreover, the presence of both ammonia and glutamate blocks USA uptake in a known [URE3] strain, so that detection of the prion is prevented, rather than its generation.

Glutamic Acid↗
PubMed2006

Ageing in yeast does not enhance prion generation.

The yeast prions [URE3] and [PSI(+)] are self-propagating amyloids of Ure2p and Sup35p, respectively. The analogous transmissible spongiform encephalopathies of mammals and other amyloidoses are largely diseases of later life. From normal strains lacking the prions, we isolated old cells and measured the frequency of de novo [URE3] and [PSI(+)] prion generation. We find no evidence that ageing of yeast increases the frequency of prion occurrence.

Amyloid↗
PubMed2006

How to find a prion: [URE3], [PSI+] and [beta].

Infectious proteins (prions) in yeast or other microorganisms can be identified by genetic methods of rather general applicability. Infection in yeast means transfer by cytoplasmic mixing (cytoduction), a property of all non-chromosomal genetic elements whether plasmids, viruses, or prions. Prions can be diagnosed by reversible curability, increased occurrence when the corresponding protein is overproduced, a requirement for the gene for the corresponding protein for propagation, and, in some cases, similarity of phenotype of: (a) mutations in the gene for the protein and (b) the presence of the prion. This approach is illustrated with [URE3], an amyloid-based prion of the regulator of nitrogen catabolism, Ure2p and [PSI(+)] as a prion of the translation termination factor Sup35p. The prion concept is not limited to infectious amyloids, but includes proteins whose active form is necessary for the activation of the inactive precursor. We detail methods used in studies of [URE3] and [beta], a self-activating protease, some of which are of broad application.

Aspartic Acid↗
PubMed2005

Primary sequence independence for prion formation.

Many proteins can adopt self-propagating beta-sheet-rich structures, termed amyloid fibrils. The [URE3] and [PSI+] prions of Saccharomyces cerevisiae are infectious amyloid forms of the proteins Ure2p and Sup35p, respectively. Ure2p forms prions primarily as a result of its sequence composition, as versions of Ure2p with the prion domain amino acids shuffled are still able to form prions. Here we show that prion induction by both Ure2p and Ure2-21p, one of the scrambled versions of Ure2p, is clearly dependent on the length of the inducing fragment. For Ure2-21p, no single sequence is found in all of the inducing fragments, highlighting the sequence independence of prion formation. Furthermore, the sequence of the Sup35p prion domain can also be randomized without blocking prion formation. Indeed, a single shuffled sequence could give rise to several prion variants. These results suggest that [PSI+] formation is driven primarily by the amino acid composition of the Sup35p prion domain, and that the Sup35p oligopeptide repeats are not required for prion maintenance.

Amino Acid Sequence↗
PubMed2005

Yeast prions [URE3] and [PSI+] are diseases.

Viruses, plasmids, and prions can spread in nature despite being a burden to their hosts. Because a prion arises de novo in more than one in 10(6) yeast cells and spreads to all offspring in meiosis, its absence in wild strains would imply that it has a net deleterious effect on its host. Among 70 wild Saccharomyces strains, we found the [PIN+] prion in 11 strains, but the [URE3] and [PSI+] prions were uniformly absent. In contrast, the "selfish" 2mu DNA was in 38 wild strains and the selfish RNA replicons L-BC, 20S, and 23S were found in 8, 14, and 1 strains, respectively. The absence of [URE3] and [PSI+] in wild strains indicates that each prion has a net deleterious effect on its host.

Glutathione Peroxidase↗
PubMed2005

Is the prion domain of soluble Ure2p unstructured?

The [URE3] prion is a self-propagating amyloid form of the Ure2 protein of Saccharomyces cerevisiae. Deletions in the C-terminal nitrogen regulation domain of Ure2p increase the frequency with which the N-terminal prion domain polymerizes into the prion form, suggesting that the C-terminus stabilizes the prion domain or that the structured C-terminal region sterically impairs amyloid formation. We find by in vivo two-hybrid analysis no evidence of interaction of prion domain and C-terminal domain. Furthermore, surface plasmon resonance spectrometry shows no evidence of interaction of prion domain and C-terminal domain, and cleavage at a specific site between the domains frees the two fragments. Our NMR analysis indicates that most residues of the prion domain are in fact disordered in the soluble form of Ure2p. Deleting the tether holding the C-terminal structured region to the amyloid core does not impair prion formation, arguing against steric impairment of amyloid formation. These results suggest that the N-terminal prion domain is unstructured in the soluble protein and does not have a specific interaction with the C-terminus.

Amino Acid Sequence↗
PubMed2005

Filaments of the Ure2p prion protein have a cross-beta core structure.

Formation of filaments by the Ure2 protein constitutes the molecular mechanism of the [URE3] prion in yeast. According to the "amyloid backbone" model, the N-terminal asparagine-rich domains of Ure2p polymerize to form an amyloid core fibril that is surrounded by C-terminal domains in their native conformation. Protease resistance and Congo Red binding as well as beta-sheet content detected by spectroscopy-all markers for amyloid-have supported this model, as has the close resemblance between 40 A N-domain fibrils and the fibrillar core of intact Ure2p filaments visualized by cryo-electron microscopy and scanning transmission electron microscopy. Here, we present electron diffraction and X-ray diffraction data from filaments of Ure2p, of N-domains alone, of fragments thereof, and of an N-domain-containing fusion protein that demonstrate in each case the 4.7A reflection that is typical for cross-beta structure and highly indicative of amyloid. This reflection was observed for specimens prepared by air-drying with and without sucrose embedding. To confirm that the corresponding structure is not an artifact of air-drying, the reflection was also demonstrated for specimens preserved in vitreous ice. Local area electron diffraction and X-ray diffraction from partially aligned specimens showed that the 4.7A reflection is meridional and therefore the underlying structure is cross-beta.

Glutathione Peroxidase↗
PubMed2005

Prion domains: sequences, structures and interactions.

Mammalian and most fungal infectious proteins (also known as prions) are self-propagating amyloid, a filamentous beta-sheet structure. A prion domain determines the infectious properties of a protein by forming the core of the amyloid. We compare the properties of known prion domains and their interactions with the remainder of the protein and with chaperones. Ure2p and Sup35p, two yeast prion proteins, can still form prions when the prion domains are shuffled, indicating a parallel in-register beta-sheet structure.

Amyloid↗
PubMed2004

The N-terminal prion domain of Ure2p converts from an unfolded to a thermally resistant conformation upon filament formation.

According to the "amyloid backbone" model of Ure2p prionogenesis, the N-terminal domain of Ure2p polymerizes to form an amyloid filament backbone surrounded by the C-terminal domains. The latter domains retain their native glutathione-S-transferase (GST)-like fold but are sterically inactivated from their regulatory role in nitrogen catabolism. We have tested this model by differential scanning calorimetry of soluble and filamentous Ure2p and of soluble C-terminal domains, combined with electron microscopy. As predicted, the C-terminal domains respond to thermal perturbation identically in all three states, exhibiting a single endotherm at 76 degrees C. In contrast, no thermal signal was associated with the N-terminal domains: in the soluble state of Ure2p, because they are unfolded; in the filamentous state, because their robust amyloid conformation resists heating to 100 degrees C.

Calorimetry, Differential Scanning↗
PubMed2004

A model for Ure2p prion filaments and other amyloids: the parallel superpleated beta-structure.

In its prion form, Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae, polymerizes into filaments whereby its C-terminal regulatory domain is inactivated but retains its native fold. The filament has an amyloid fibril backbone formed by the Asn-rich, N-terminal, "prion" domain. The prion domain is also capable of forming fibrils when alone or when fused to other proteins. We have developed a model for the fibril that we call a parallel superpleated beta-structure. In this model, the prion domain is divided into nine seven-residue segments, each with a four-residue strand and a three-residue turn, that zig-zag in a planar serpentine arrangement. Serpentines are stacked axially, in register, generating an array of parallel beta-sheets, with a small and potentially variable left-hand twist. The interior of the filament is mostly stabilized not by packing of apolar side chains but by H-bond networks generated by the stacking of Asn side chains: charged residues are excluded. The model is consistent with current biophysical, biochemical, and structural data (notably, mass-per-unit-length measurements by scanning transmission electron microscopy that gave one subunit rise per 0.47 nm) and is readily adaptable to other amyloids, for instance the core of Sup35p filaments and glutamine expansions in huntingtin.

Amino Acid Sequence↗
PubMed2004

[URE3] prion propagation is abolished by a mutation of the primary cytosolic Hsp70 of budding yeast.

[URE3] and [PSI(+)] are infectious protein forms of the Saccharomyces cerevisiae Ure2p and Sup35p, respectively. We isolated an allele of SSA2, the primary cytosolic Hsp70, in a screen for mutants unable to maintain [URE3]. Designated ssa2-10, the mutation results in a leucine substitution for proline 395, a conserved residue of the peptide-binding domain. This allele also unexpectedly destabilizes [URE3] in newly formed heterozygotes: [URE3] is either absent in heterozygotes formed by crossing wild-type [URE3] cells with ssa2-10 mutants, or present and fully stable. SSA2 deletion mutants are weakly capable of maintaining [URE3]. The ssa2-10 allele is compatible with propagation of [PSI(+)]. However, in combination with a deletion of SSA1, ssa2-10 eliminates the nonsense-suppression phenotype of [PSI(+)] cells.

Cloning, Molecular↗
PubMed2004

Prions of yeast fail to elicit a transcriptional response.

Amyloid deposits are associated with numerous human diseases. The [URE3] prion of Saccharomyces is an infectious, inactive, amyloid form of the Ure2p protein. Despite the presence of large prion aggregates in [URE3] yeast, the only apparent phenotypes associated with the prion are attributable to loss of Ure2p function. We used cDNA microarrays to look for genes in yeast that are differentially expressed in the presence of the [URE3] prion and which might act to mitigate the detrimental effects of the prion aggregates. On comparing [URE3] vs. ure2 yeast, we were surprised to find that the only expression changes detected were attributable to the low level of residual Ure2p activity in the [URE3] cells. Interestingly, in addition to repressing the activity of genes required for utilization of poor nitrogen sources when yeast are grown in the presence of a good nitrogen source, Ure2p appears to be involved in stimulating some of these same genes in the absence of a good nitrogen source.

Culture Media↗
PubMed2004

Scrambled prion domains form prions and amyloid.

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The amino-terminal prion domain of Ure2p is necessary and sufficient for prion formation and has a high glutamine (Q) and asparagine (N) content. Such Q/N-rich domains are found in two other yeast prion proteins, Sup35p and Rnq1p, although none of the many other yeast Q/N-rich domain proteins have yet been found to be prions. To examine the role of amino acid sequence composition in prion formation, we used Ure2p as a model system and generated five Ure2p variants in which the order of the amino acids in the prion domain was randomly shuffled while keeping the amino acid composition and C-terminal domain unchanged. Surprisingly, all five formed prions in vivo, with a range of frequencies and stabilities, and the prion domains of all five readily formed amyloid fibers in vitro. Although it is unclear whether other amyloid-forming proteins would be equally resistant to scrambling, this result demonstrates that [URE3] formation is driven primarily by amino acid composition, largely independent of primary sequence.

Amino Acid Sequence↗
PubMed2004

Prion genetics: new rules for a new kind of gene.

Just as nucleic acids can carry out enzymatic reactions, proteins can be genes. These heritable infectious proteins (prions) follow unique genetic rules that enable their identification: reversible curing, inducible "spontaneous generation," and phenotype surprises. Most prions are based on self-propagating amyloids, depend heavily on chaperones, show strain phenomena and, like other infectious elements, show species barriers to transmission. A recently identified prion is based on obligatory self-activation of an enzyme in trans. Although prions can be detrimental, they may also be beneficial to their hosts.

Amyloid↗