Recombinant Saccharomyces cerevisiae Copper transport protein CTR3 (CTR3)

What is the structural organization of the CTR3 protein in Saccharomyces cerevisiae?

CTR3 in Saccharomyces cerevisiae is an integral membrane protein that assembles as a trimer to form a competent copper uptake permease at the plasma membrane. This trimeric assembly is crucial for its function as a high-affinity copper transporter . The protein's structural configuration enables it to create a pore-like structure through which copper ions can be transported across the plasma membrane. Unlike its functionally redundant counterpart Ctr1, CTR3 bears little homology at the amino acid sequence level, suggesting distinct evolutionary origins despite convergent functions . Research indicates that CTR3 constitutes a fundamental module found in all eukaryotic high-affinity copper transporters identified to date, which is sufficient for copper uptake but notably lacks elements required for post-transcriptional regulation by copper.

How does CTR3 differ functionally from Ctr1 in copper transport mechanisms?

While CTR3 and Ctr1 are functionally redundant in their primary role of high-affinity copper transport into S. cerevisiae cells, they exhibit significant differences in their post-transcriptional regulation mechanisms . The key distinction lies in their response to elevated copper levels. Unlike Ctr1, which undergoes protein degradation and endocytosis when copper levels increase, CTR3 does not exhibit these regulatory responses . This suggests that while both proteins serve the same essential function of copper uptake, they are integrated into different cellular regulatory networks. The CTR3 transporter appears to maintain its presence at the plasma membrane regardless of environmental copper concentrations, which may represent an evolutionary strategy to ensure baseline copper acquisition even under fluctuating conditions.

What are the most effective experimental approaches for studying CTR3 expression and localization?

For investigating CTR3 expression and localization in Saccharomyces cerevisiae, researchers should employ a multi-faceted approach combining molecular and cellular techniques. At the transcriptional level, quantitative RT-PCR provides precise measurements of CTR3 mRNA levels under various conditions. Following established protocols, mRNA should be extracted, cDNA constructed using random oligonucleotides, and RT-PCR analyses performed with appropriate normalization controls such as actin . For protein localization studies, fluorescent protein tagging (e.g., GFP fusion) combined with confocal microscopy provides valuable insights into CTR3's cellular distribution and membrane integration. Additionally, biochemical fractionation techniques can confirm CTR3's presence in the plasma membrane fraction. To study the trimeric assembly of CTR3, cross-linking studies followed by non-denaturing gel electrophoresis can preserve and detect the native oligomeric state, while copper uptake assays using radioactive 64Cu or colorimetric detection methods can directly measure transport activity of wild-type versus mutant CTR3 variants.

How can researchers effectively distinguish between CTR3 and Ctr1 functions in copper transport studies?

Distinguishing between CTR3 and Ctr1 functions requires strategic experimental design that capitalizes on their different regulatory mechanisms. Since CTR3 and Ctr1 are functionally redundant but differentially regulated post-transcriptionally, researchers can exploit this distinction to isolate their individual contributions .

A systematic approach would involve:

• Genetic manipulation: Creating single knockout strains (Δctr1 and Δctr3) and double knockout strains (Δctr1Δctr3) supplemented with copper to assess specific phenotypes.

• Copper response assays: Exposing cells to elevated copper levels, which triggers Ctr1 degradation but not CTR3 degradation, creating a window where CTR3 function can be studied in isolation .

• Promoter replacement: Placing CTR3 or Ctr1 under the control of inducible promoters (e.g., GAL1) to control expression independently of copper levels.

• Domain swap experiments: Creating chimeric proteins with domains from both transporters to identify which regions confer specific regulatory properties.

• Quantitative copper uptake measurements: Using radioactive 64Cu trace experiments under conditions where one transporter is selectively active or inactive.

What CRISPR-based techniques are most suitable for CTR3 gene manipulation in Saccharomyces cerevisiae?

For precise CTR3 gene manipulation in S. cerevisiae, CRISPR-Cas9 based approaches offer powerful tools for both gene knockout and precise editing. The CRISPR D-BUGS protocol mentioned in the Sc2.0 project context can be adapted for identifying and modifying defective loci in CTR3 studies .

When implementing CRISPR for CTR3 manipulation, researchers should consider:

• gRNA design: Target sequences should be selected for high specificity to the CTR3 locus while minimizing off-target effects. Multiple computational tools are available for gRNA design optimized for S. cerevisiae.

• Delivery method: Plasmid-based expression of Cas9 and gRNA components works efficiently in yeast. The commonly used approach involves a plasmid containing the Cas9 gene under a constitutive or inducible promoter and another plasmid or cassette expressing the gRNA.

• Repair template design: For precise edits (point mutations, insertions, or deletions), researchers should design repair templates with homology arms of 40-60 bp flanking the desired mutation site.

• Transformation and selection: Standard lithium acetate transformation protocols can be used, followed by selection for markers included in the CRISPR plasmids.

• Verification strategies: Successful edits should be confirmed by sequencing and functional assays to ensure that CTR3 function has been modified as intended.

What expression systems are optimal for recombinant CTR3 production in heterologous hosts?

For optimal recombinant CTR3 production in heterologous hosts, researchers should consider several expression systems tailored to the specific research objectives. When expressing yeast membrane proteins like CTR3, the choice of expression system significantly impacts protein folding, membrane insertion, and functional activity.

The following table summarizes key expression systems for recombinant CTR3 production:

For fundamental studies, using S. cerevisiae itself as an expression host offers advantages, as it provides the native cellular machinery for proper folding and assembly of the trimeric CTR3 complex . In this case, the TDH1 promoter system (as used for XYL1 and XYL2 in other S. cerevisiae studies) provides strong constitutive expression . Each system requires optimization of codons, signal sequences, and purification tags specific to CTR3's requirements as a trimeric membrane protein.

What are the key considerations for creating CTR3 fusion proteins that maintain transport functionality?

Creating functional CTR3 fusion proteins requires careful design considerations to preserve the protein's trimeric assembly and transport activity. Since CTR3 is an integral membrane protein that forms a trimer at the plasma membrane , fusion tags must be positioned to avoid disrupting these critical structural features.

Key considerations include:

• Tag position: For membrane proteins like CTR3, N-terminal tags are generally preferred over C-terminal tags, as the C-terminus may contain signals important for membrane targeting or protein-protein interactions.

• Flexible linkers: Incorporating glycine-serine linkers (e.g., GGGGS) between CTR3 and the fusion tag can minimize steric hindrance and allow both components to fold properly.

• Tag selection: For localization studies, fluorescent proteins like GFP or mCherry are valuable, while epitope tags (HA, FLAG, His6) facilitate purification and Western blot detection. Consider smaller tags for functional studies to minimize disruption.

• Verification methods: Functional validation is critical. Researchers should confirm:

  • Proper membrane localization using microscopy or fractionation

  • Trimeric assembly using blue native PAGE or crosslinking approaches

  • Transport activity using copper uptake assays

  • Complementation of growth defects in ctr1Δctr3Δ strains

• Inducible expression: Using controllable promoters allows titration of fusion protein levels, helping to distinguish between functional defects and toxicity from overexpression.

How can researchers effectively monitor CTR3 transcriptional regulation in response to varying copper levels?

Monitoring CTR3 transcriptional regulation in response to copper levels requires a combination of molecular techniques. Based on research approaches in yeast gene expression studies, an effective methodology would include:

• Real-time quantitative PCR (RT-qPCR): This approach allows precise measurement of CTR3 mRNA levels under various conditions. Researchers should extract mRNA following established protocols, construct cDNA using random oligonucleotides, and perform RT-PCR analyses using appropriate instruments. Actin can serve as a normalization control, and a standard curve using genomic DNA should be established for quantification .

• Promoter-reporter fusion assays: Constructing fusions between the CTR3 promoter and reporter genes such as GFP, β-galactosidase, or luciferase enables real-time monitoring of transcriptional activity in living cells under varying copper concentrations.

• Chromatin immunoprecipitation (ChIP): To understand the transcription factors involved in CTR3 regulation, ChIP assays can identify protein-DNA interactions at the CTR3 promoter under different copper conditions.

• RNA-seq analysis: This approach provides a genome-wide view of transcriptional changes, allowing researchers to place CTR3 regulation within the broader context of the cellular response to copper availability.

When designing experiments, researchers should carefully control copper concentrations in media, considering both copper-depleted conditions (using chelators) and copper-replete conditions with precise supplementation.

How does post-transcriptional regulation of CTR3 differ from other copper transporters?

The post-transcriptional regulation of CTR3 displays distinctive characteristics that set it apart from other copper transporters in S. cerevisiae, most notably its functionally redundant counterpart, Ctr1. Based on the research findings, CTR3 and CTR1 exhibit similar transcriptional regulation in response to copper availability, but their post-transcriptional regulation mechanisms are markedly different .

The most significant distinction is that CTR3 does not undergo protein degradation or endocytosis in response to elevated copper levels, unlike Ctr1 . This fundamental difference suggests that while both proteins serve as high-affinity copper transporters, they are integrated into different cellular regulatory networks. The persistence of CTR3 at the plasma membrane regardless of environmental copper concentrations indicates that this transporter may serve as a constitutive component of the copper uptake machinery.

This regulatory distinction has important implications:

• CTR3 likely lacks the specific protein domains or motifs that target Ctr1 for degradation or endocytosis in high-copper conditions.

• The differential regulation may represent an evolutionary strategy to maintain baseline copper uptake capacity even under conditions that trigger downregulation of other transport systems.

• CTR3 may function as a "backbone" copper transport system that ensures essential copper uptake, while Ctr1 serves as a more dynamically regulated system that responds to acute changes in copper availability.

What methodological approaches are most effective for studying the trimeric assembly of CTR3?

Studying the trimeric assembly of CTR3 requires specialized methodological approaches that preserve the native oligomeric state while yielding structural and functional insights. Based on the understanding that CTR3 forms a trimer at the plasma membrane to create a functional copper transport permease , researchers should consider the following methodological approaches:

• Crosslinking studies: Chemical crosslinking with membrane-permeable reagents like DSP (dithiobis(succinimidyl propionate)) or formaldehyde can capture the trimeric state by covalently linking adjacent subunits. Following crosslinking, SDS-PAGE and immunoblotting can reveal the oligomeric species.

• Blue Native PAGE (BN-PAGE): This non-denaturing electrophoresis technique preserves native protein complexes and can distinguish between monomeric, dimeric, and trimeric forms of membrane proteins like CTR3.

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS): This approach allows determination of the absolute molecular weight of membrane protein complexes in detergent solutions, providing information about the oligomeric state.

• Single-particle cryo-electron microscopy: For detailed structural analysis, cryo-EM can resolve the structure of membrane protein complexes like the CTR3 trimer, revealing the arrangement of subunits and potential copper-binding sites.

• Förster resonance energy transfer (FRET): By tagging different populations of CTR3 with appropriate fluorophore pairs, FRET can detect close association between subunits in living cells, confirming oligomerization under physiological conditions.

• Mutagenesis of putative interface residues: Systematic mutation of amino acids predicted to be at subunit interfaces, followed by functional assays and oligomerization analysis, can identify residues critical for trimerization.

What role does CTR3 play in copper homeostasis during environmental stress responses in yeast?

CTR3's role in copper homeostasis during environmental stress responses in yeast represents an important area of investigation. While the search results don't directly address CTR3's specific function during stress conditions, we can integrate knowledge about copper transport with information about yeast stress responses to develop a research framework.

S. cerevisiae responds to various environmental stresses through complex transcriptional programs involving multiple signaling pathways . During stress responses, copper homeostasis becomes particularly important as copper is an essential cofactor for enzymes involved in oxidative stress defense (like Cu/Zn superoxide dismutase) and energy metabolism (like cytochrome c oxidase).

CTR3, as a high-affinity copper transporter that is not subjected to degradation or endocytosis in response to elevated copper levels , likely plays a distinctive role during stress conditions:

• Temperature stress: During temperature fluctuations, membrane fluidity changes can affect transporter function. The HOG pathway involved in adaptation to cold stress affects membrane properties , potentially influencing CTR3 activity.

• Oxidative stress: Increased demand for copper-containing antioxidant enzymes during oxidative stress may be partially met through CTR3-mediated copper uptake. Its non-degradable nature could ensure continued copper acquisition even under stress conditions.

• Nutritional stress: Under nutrient limitation, the TORC1-Sch9 pathway regulates stress responses . This pathway may influence copper transport priorities, potentially affecting the relative contributions of CTR3 versus Ctr1.

• Osmotic stress: The HOG pathway mediates responses to osmotic stress , which may indirectly impact CTR3 function through changes in membrane tension or cellular signaling.

How can CTR3 be utilized in synthetic biology applications for metal homeostasis engineering?

CTR3's distinctive properties as a high-affinity copper transporter that maintains stable membrane presence regardless of copper levels positions it as a valuable component for synthetic biology applications focused on metal homeostasis engineering. Researchers can exploit these characteristics in several innovative ways:

• Engineered metal sensing and accumulation systems: By coupling CTR3 expression with metal-responsive promoters not native to copper regulation, synthetic circuits can be designed where CTR3 expression is triggered by other metals or environmental signals. This approach can create yeast strains with novel metal accumulation profiles for bioremediation or metal recovery applications.

• Stable copper uptake chassis: The Sc2.0 synthetic yeast genome project represents an opportunity to incorporate modified versions of CTR3 as standardized parts for consistent copper uptake. Since CTR3 lacks the post-transcriptional regulation that affects other transporters like Ctr1 , it can provide a more predictable copper uptake component in synthetic systems.

• Copper-dependent enzyme production: Many industrial enzymes require copper as a cofactor. Engineering strains with optimized CTR3 expression could enhance the production of copper-dependent enzymes for biocatalysis applications.

• Metabolic engineering: Copper is essential for respiratory growth in yeast. Optimizing CTR3 expression could improve growth on non-fermentable carbon sources, potentially enhancing production of certain metabolites in industrial fermentations.

• Biosensor development: CTR3 could be incorporated into synthetic circuits as part of copper-sensing systems, where its activity is coupled to reporter gene expression to create whole-cell biosensors for environmental copper detection.

What methodologies are most effective for studying CTR3's role in copper-dependent enzyme production?

Investigating CTR3's role in copper-dependent enzyme production requires a multi-faceted experimental approach that links copper transport to downstream metabolic processes. Effective methodologies should focus on establishing clear connections between CTR3 function, intracellular copper availability, and the activity of copper-dependent enzymes.

A comprehensive experimental strategy would include:

• Genetic manipulation approaches:

  • Create a series of strains with varying CTR3 expression levels (deletion, native, and overexpression)

  • Generate strains with CTR3 under inducible promoters to modulate expression during specific growth phases

  • Construct strains expressing CTR3 variants with altered transport kinetics

  • Compare with Ctr1 variants to leverage their different post-transcriptional regulation

• Enzymatic activity assays:

  • Measure the activity of copper-dependent enzymes such as Cu/Zn superoxide dismutase (Sod1) and cytochrome c oxidase

  • Correlate enzyme activity with CTR3 expression levels and intracellular copper content

  • Perform time-course studies following copper addition to copper-starved cells

• Intracellular copper tracking:

  • Use copper-specific fluorescent probes to monitor intracellular copper distribution

  • Employ radioactive 64Cu to quantitatively track copper uptake and distribution

  • Fractionate cells to determine copper allocation to different compartments and copper-dependent enzymes

• Systems biology approaches:

  • Perform transcriptomics to identify genes co-regulated with CTR3 under varying copper conditions

  • Use proteomics to monitor changes in the abundance of copper-dependent enzymes

  • Apply metabolomics to assess the impact on metabolic pathways dependent on copper-containing enzymes

How can researchers integrate CTR3 studies with the Sc2.0 synthetic yeast genome project?

The Sc2.0 global consortium project, which aims to construct a synthetic Saccharomyces cerevisiae genome , presents unique opportunities for advancing CTR3 research through integration with synthetic genomics approaches. Researchers interested in combining CTR3 studies with the Sc2.0 project should consider several strategic approaches:

• Standardized CTR3 variants in synthetic chromosomes:

  • Design optimized CTR3 coding sequences following Sc2.0 design principles, including codon optimization and removal of destabilizing elements

  • Incorporate systematically varied promoters and terminators to create a library of CTR3 expression variants

  • Include synXVI-compatible loxPsym sites for later SCRaMbLE-based evolution of novel CTR3 regulation

• Application of CRISPR D-BUGS methodology:

  • Utilize the CRISPR D-BUGS protocol developed in the Sc2.0 project to systematically identify and debug defective loci affecting CTR3 function

  • Implement iterative design-build-test-learn cycles to optimize CTR3 performance in synthetic genome contexts

  • Apply lessons learned from synXVI debugging to anticipate and prevent integration issues when modifying CTR3 in synthetic genomes

• Systematic domain analysis through synthetic biology:

  • Design modular, swappable domains in CTR3 based on the principle of chunk and megachunk termini used in Sc2.0

  • Create a series of chimeric constructs combining domains from CTR3 and other transporters to identify functional regions

  • Implement high-throughput phenotypic analysis to characterize the function of synthetic CTR3 variants

• Integration with tRNA neochromosome:

  • Explore optimization of CTR3 expression through specialized tRNA availability on the Sc2.0 tRNA neochromosome

  • Investigate whether CTR3 codon usage can be optimized for expression using the synthetic tRNA system