Recombinant Saccharomyces paradoxus Protein URE2 (URE2)

What is the structure and function of S. paradoxus URE2 protein?

The URE2 protein from Saccharomyces paradoxus shares structural similarity with its well-studied ortholog in S. cerevisiae. Like other Ure2 proteins, it consists of two primary domains: an N-terminal prion domain (approximately the first 65-93 amino acids) and a C-terminal functional domain responsible for nitrogen catabolism regulation. The C-terminal domain has a structure similar to glutathione-S-transferase superfamily members, while the prion domain enables the protein to form self-propagating amyloid structures under certain conditions .

Functionally, S. paradoxus URE2 serves as a key regulator in nitrogen catabolite repression, which allows the yeast to preferentially utilize good nitrogen sources over poor ones. When expressed in S. cerevisiae strains lacking their native URE2, the S. paradoxus protein can fully complement this function, demonstrating conservation of the regulatory mechanism .

How does the prion-forming ability of S. paradoxus URE2 compare to other yeast species?

The prion-forming ability of URE2 varies considerably across yeast species, with S. paradoxus URE2 demonstrating strong prion capabilities. Studies have shown that wild S. paradoxus strains can be infected with and propagate the [URE3] prion, suggesting that the prion mechanisms are conserved between S. paradoxus and S. cerevisiae .

When comparing across multiple species, the ability to form prions correlates with specific sequences in the N-terminal domain. For example, both S. paradoxus and S. uvarum URE2 proteins maintain prion-forming capabilities, while the URE2 from Candida glabrata cannot form [URE3] despite having a conserved sequence that was thought to be important for prion formation . This finding challenges earlier hypotheses about which specific sequences are necessary for prion formation.

The prion domain of S. paradoxus (PrD Sp) is actually more efficient at [URE3] induction than its S. cerevisiae counterpart (PrD Sc), possibly due to specific amino acid differences .

What experimental systems are commonly used to study S. paradoxus URE2?

Several experimental systems have been developed to study S. paradoxus URE2:

• Complementation assays: Testing the ability of S. paradoxus URE2 to complement S. cerevisiae ure2Δ mutants provides insights into functional conservation of the nitrogen regulation role .

• Prion induction and propagation: The [URE3] prion state can be monitored using reporter systems based on the DAL5 promoter controlling ADE2 gene expression. In [ure-o] cells (non-prion state), cells are Ade-, while [URE3] cells are Ade+ because active Ure2p prevents transcription from the DAL5 promoter .

• Cross-species prion transmission: Researchers can study whether prion states can be transmitted between URE2 proteins from different species, providing insights into species barriers and prion biology .

• Genetic crosses: Meiotic crosses between different yeast strains have been used to determine whether wild S. paradoxus can propagate [URE3] prions .

What molecular mechanisms influence cross-species prion transmission with S. paradoxus URE2?

Cross-species prion transmission between S. cerevisiae and S. paradoxus URE2 proteins demonstrates interesting patterns that inform our understanding of prion biology. Research shows that [URE3] prions can be efficiently transmitted from S. cerevisiae into S. paradoxus ure2Δ cells expressing Ure2p from species within Saccharomyces .

The molecular factors influencing transmission include:

• Sequence similarity: Higher sequence similarity in the prion domains correlates with more efficient cross-species transmission. The close evolutionary relationship between S. cerevisiae and S. paradoxus likely explains their compatible prion transmission .

• Conserved structural elements: Residues 11-39 in the S. cerevisiae prion domain are necessary for inactivating interactions with full-length Ure2p. This region is highly conserved among many yeasts despite sequence divergence in other parts of the N-terminal domains .

• Species barrier mechanisms: The [URE3] prion of S. cerevisiae cannot seed self-propagating inactivation of URE2 proteins from more distantly related yeasts like Candida albicans, despite functional conservation of the C-terminal domain .

When [Sc URE3] strains are crossed with wild-type strains, the resulting diploids can maintain the prion state, indicating transfer of the prion conformation between proteins. No species barrier exists between S. cerevisiae and S. uvarum proteins, allowing prion inactivation to be transmitted regardless of which protein was initially affected .

How does amino acid sequence conservation in the prion domain relate to function?

The relationship between sequence conservation and prion-forming ability is complex and sometimes counterintuitive. Research has revealed several key insights:

• Differential evolutionary pressure: The C-terminal functional domain and N-terminal prion domain have diverged separately, with the functional domain showing greater conservation across species, reflecting stronger selective pressure on nitrogen regulation function than on prion formation .

• Conserved sequences not required for prion formation: Contrary to earlier hypotheses, conservation of amino acid sequences within the prion domain does not necessarily correlate with prion-forming ability. For example, C. albicans Ure2p can form [URE3] prions in S. cerevisiae despite lacking the conserved sequence, while C. glabrata Ure2p cannot form [URE3] despite having the conserved sequence .

• Functional relevance of prion domains: The absence of prion domains in S. pombe Ure2p homologs correlates with the absence of a specific structural region (the "clip" or "cap" region, amino acids 267-295) in the functional domain. This suggests a potential functional relationship between these regions beyond prion formation .

• Asparagine tracks: Expansion or contraction of asparagine-rich regions affects aggregation propensity. S. uvarum URE2 has an expanded asparagine track, while S. paradoxus has a deletion of seven asparagine residues at the same position .

These findings suggest that the conservation of certain sequences in the prion domain may serve functions beyond maintaining prion-forming ability.

What methods are most effective for studying the prion properties of S. paradoxus URE2?

Several sophisticated methods have proven valuable for investigating prion properties of S. paradoxus URE2:

• Reporter systems: The DAL5 promoter controlling ADE2 expression provides a visual indicator of Ure2p activity. [ure-o] cells are Ade-, while [URE3] cells are Ade+ because active Ure2p prevents transcription from the DAL5 promoter .

• Domain swapping experiments: Exchanging prion domains between species and testing prion formation helps identify critical regions for prion propagation. These experiments have demonstrated that the prion domains of S. paradoxus and S. uvarum URE2 have retained the capability to induce [URE3] in S. cerevisiae strains .

• Cytoduction: This technique allows transfer of cytoplasmic elements (including prions) without nuclear mixing, enabling researchers to study prion transmission between different strains or species. Studies using cytoduction have shown that wild S. paradoxus strains can stably maintain [URE3para] prions .

• Overexpression systems: Expressing URE2 under control of inducible promoters (such as GAL1) allows researchers to test for prion curing, induction, or complementation under controlled conditions .

• Spin label scanning: Recent research employs spin label scanning to reveal the likely locations of β-strands in the amyloid core of URE2 prions, providing structural information about the prion form .

How are recombinant S. paradoxus URE2 proteins typically produced and purified?

Production of recombinant S. paradoxus URE2 typically involves:

• Gene amplification: The URE2 gene from S. paradoxus genomic DNA can be amplified using PCR with primers designed based on sequence information. For example, researchers have used primers that target conserved regions of URE2 at different hybridization temperatures (38-49°C) followed by cloning and sequencing of the amplification products .

• Expression systems: The gene is typically cloned into expression vectors under control of inducible promoters. For studies in yeast, multicopy expression vectors with the GAL1 promoter are commonly used, allowing controlled expression by switching between glucose and galactose media . For biochemical studies requiring purified protein, bacterial expression systems using E. coli are often employed.

• Protein purification: Standard protein purification techniques are applicable, including affinity chromatography (if tagged versions are used), ion exchange chromatography, and size exclusion chromatography. The specific properties of URE2 (such as its tendency to form aggregates under certain conditions) should be considered when designing purification protocols.

• Quality control: Ensuring proper folding and activity of the purified protein is essential, especially when studying prion properties that depend on specific conformational states. Functional assays, such as testing nitrogen regulation activity in complementation experiments, provide verification of proper protein function .

What are effective methods for studying the structural transitions of URE2 from soluble to prion forms?

Understanding the structural transitions of URE2 from its soluble to prion forms requires specialized techniques:

• Amyloid-specific dyes: Thioflavin T fluorescence and Congo Red binding assays can monitor the formation of amyloid structures characteristic of prion forms.

• Electron microscopy: Negative staining and electron microscopy allow visualization of amyloid fibrils formed by the prion domain.

• Protein misfolding cyclic amplification (PMCA): This technique can amplify small amounts of prion seeds, facilitating studies of prion propagation and species barriers.

• Protease resistance assays: The prion form of URE2 typically shows increased resistance to proteolytic digestion compared to the soluble form, providing a biochemical method to distinguish between conformational states.

• Spin labeling and EPR spectroscopy: Recent research has employed spin label scanning to identify the β-strand arrangements in amyloid fibrils formed by URE2 . This technique provides valuable structural information about the prion conformation.

• Fluorescence microscopy with fusion proteins: GFP-URE2 fusions have been used to visualize aggregation states in vivo. For example, studies have shown that residues 5-47 of the S. cerevisiae prion domain, when overexpressed as a fusion with GFP, can cure the [URE3] prion .

What does the conservation pattern of URE2 reveal about its evolutionary history?

The evolutionary analysis of URE2 across fungal species reveals several important insights:

• Differential selective pressure: The C-terminal functional domain of URE2 is more highly conserved than the N-terminal prion domain, reflecting stronger evolutionary constraints on the nitrogen regulation function . This suggests that the primary selective pressure has been on maintaining metabolic function rather than prion-forming ability.

• Taxonomic distribution: The URE2 gene is not universally present in all fungi. For example, data from the Genolevure project indicates that URE2 is not systematically present in all hemiascomycetes genomes . This suggests that the gene may have been lost in some lineages or gained in others through evolutionary processes.

• Correlation between domains: The presence or absence of the prion domain correlates with structural features in the functional domain. For instance, S. pombe homologs lack both the prion domain and a specific structural region (the "clip" or "cap" region, amino acids 267-295) in the functional domain . This correlation suggests a potential functional relationship between these regions.

• Functional conservation: Despite sequence divergence, the nitrogen regulation function of URE2 is well-conserved across species. URE2 genes from S. paradoxus, S. uvarum, K. lactis, and C. albicans can all complement URE2 deletion in S. cerevisiae , demonstrating functional conservation despite sequence changes.

The evolutionary pattern suggests that URE2 evolved primarily to regulate nitrogen metabolism, while its prion-forming ability may be either a secondary adaptation or a coincidental property arising from the protein's sequence and structure.

How does the S. paradoxus URE2 prion domain compare to other yeast species at the sequence level?

Sequence comparison of the prion domains across different yeast species reveals interesting patterns:

• Asparagine content variations: The prion domains of different species show variations in their asparagine content and distribution. For example, S. uvarum URE2 has an expansion of the asparagine track, which generally favors protein aggregation, while S. paradoxus has a deletion of seven asparagine residues at the same position .

• Key functional regions: Residues 11-39 of the S. cerevisiae prion domain are necessary for inactivating interactions with full-length Ure2p. This region is highly conserved among many yeasts despite sequence divergence in other parts of the N-terminal domains .

• Specific amino acid changes: Specific substitutions may significantly impact prion-forming ability. For instance, the N79D change (asparagine to aspartate) found in some species might affect prion induction, as similar mutations have been shown to dramatically impact [PSI+] prion induction in other proteins .

• Complete absence in some species: Some distant relatives, such as S. pombe, have URE2 homologs that completely lack the prion domain . This suggests that the prion domain is not essential for the fundamental nitrogen regulation function.

These sequence variations provide valuable natural experiments for understanding which sequence features are critical for prion formation and propagation.