Recombinant Schizosaccharomyces pombe Copper transport protein ctr5 (ctr5)

Why can't Ctr5 function independently in copper transport?

Unlike other members of the Ctr1 family (such as human and Saccharomyces cerevisiae Ctr1 proteins), Ctr5 cannot function independently because it requires Ctr4 to form a functional heteromeric complex. This interdependence stems from:

• Mutual requirement for maturation through the secretory pathway

• Interdependence for localization to the plasma membrane

• Necessity for proper complex assembly for copper transport activity

Without Ctr4, Ctr5 becomes trapped within the secretory pathway and fails to reach the plasma membrane where copper transport occurs .

How can researchers experimentally determine the stoichiometry of the Ctr4-Ctr5 complex?

Bimolecular fluorescence complementation (BiFC) assays have been instrumental in determining the stoichiometry of the Ctr4-Ctr5 complex. Research has shown that a functional high-affinity copper uptake system requires a specific stoichiometric assembly of two Ctr4 molecules with one Ctr5 molecule. The methodology involves:

• Tagging Ctr4 and Ctr5 with complementary fragments of a fluorescent protein

• Co-expressing these constructs in vivo

• Analyzing fluorescence reconstitution to determine protein-protein interactions

• Using systematic mutation of interaction domains to confirm specific assembly patterns

This approach revealed that the assembly of a functional heterotrimeric complex on the cell surface requires a 2:1 ratio of Ctr4 to Ctr5 molecules .

What methods are effective for studying the trafficking of Ctr5 to the plasma membrane?

Several complementary approaches have proven effective:

These approaches revealed that the carboxyl-terminal domain (CTD) of Ctr5 plays a crucial role in trafficking of the complex to the cell surface, as demonstrated by the successful trafficking of Ctr445 chimeric protein containing the Ctr5 CTD .

What is the relative contribution of the Met-X₃-Met motif in Ctr5 to copper transport activity?

The Met-X₃-Met motif in transmembrane domain 2 (TMD2) of Ctr5 is dispensable for the functionality of the Ctr4-Ctr5 complex, unlike the same motif in Ctr4. Experimental evidence supporting this includes:

• Mutagenesis studies where Met130 and Met134 in Ctr5 were substituted with alanine residues (CTR5-M130/134A)

• Functional complementation assays in S. cerevisiae ctr1Δctr3Δ mutants showing that the Ctr4-Ctr5(M130/134A) complex remains functional

• Growth assays in Sch. pombe demonstrating that the Met-X₃-Met motif mutations in Ctr5 had no effect on growth in non-fermentable carbon sources (YES-EG)

This suggests that while Ctr5 is essential for complex formation and trafficking, it does not directly participate in the copper transport mechanism through its Met-X₃-Met motif .

How does the C-terminal domain of Ctr5 contribute to the function of the Ctr4-Ctr5 complex?

The C-terminal domain (CTD) of Ctr5 plays a crucial role in the regulation of trafficking of the copper transport complex to the cell surface. This was determined through chimeric protein studies that revealed:

• Chimeric proteins containing the Ctr4 central domain and Ctr5 CTD (such as Ctr445) are functional and can reach the plasma membrane

• The CTD of Ctr4 appears to inhibit the delivery of the protein to the cell surface in Sch. pombe

• Substitution of the Ctr4 CTD with the Ctr5 CTD leads to proper localization to the cell surface

• The Ctr445 chimera can complement the growth defects of ctr4Δctr5Δ Sch. pombe strains and reaches the plasma membrane without requiring any accessory protein

These findings indicate that the Ctr5 CTD contains trafficking signals that are essential for the proper localization of the copper transport complex .

How is Ctr5 expression regulated during meiosis?

Ctr5 exhibits a distinct expression pattern during meiosis:

• It is expressed throughout the entire meiotic process, unlike Ctr4 which is primarily expressed in early meiosis

• Its expression increases during middle- and late-phase meiosis

• The regulation of ctr6+ gene expression involves two distinct regulators: Cuf1 and Mei4

• Under low copper conditions, Ctr4 and Ctr5 initially co-localize at the plasma membrane shortly after meiotic induction

• After meiotic divisions, Ctr4 and Ctr5 show differential localization patterns: Ctr4 and Ctr5 disappear from the cell surface, while Ctr6 undergoes intracellular re-location to co-localize with the forespore membrane

These temporal and spatial expression patterns suggest specialized roles for copper transporters during different stages of meiosis .

What methods are used to study Ctr5 expression under varying copper conditions?

Researchers employ multiple complementary approaches:

These studies have revealed that Ctr5 expression is induced under copper-deficient conditions, regulated by the copper-sensing transcription factor Cuf1. The Ctr4-Ctr5 complex is also post-transcriptionally regulated by copper, with both proteins being internalized in response to high copper concentrations and recycled back to the cell surface when copper availability diminishes .

What expression systems are optimal for producing recombinant Ctr5 protein?

E. coli has been successfully used as an expression system for recombinant Ctr5 production:

• The full-length Ctr5 protein (amino acids 1-173) can be expressed with an N-terminal His tag

• The recombinant protein is typically purified to >90% purity as determined by SDS-PAGE

• The protein is often provided as a lyophilized powder

• Recommended storage conditions include -20°C/-80°C for long-term storage, with aliquoting to avoid repeated freeze-thaw cycles

• For reconstitution, deionized sterile water is used to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol for long-term storage

What experimental approaches best determine the functional integrity of recombinant Ctr5?

To verify that recombinant Ctr5 maintains its functional properties, researchers can employ several techniques:

• Complementation assays in Sch. pombe ctr5Δ mutants to test biological activity

• Co-expression with Ctr4 to examine complex formation capacity

• In vitro copper binding assays to assess metal coordination properties

• Circular dichroism spectroscopy to verify proper secondary structure formation

• Limited proteolysis to evaluate protein folding

• Structural analyses through techniques such as X-ray crystallography or cryo-electron microscopy when combined with Ctr4

These approaches provide complementary information about both structural integrity and functional capacity of the recombinant protein .

How can chimeric proteins between Ctr4 and Ctr5 be designed to study domain-specific functions?

Designing functional chimeric proteins requires careful consideration of domain boundaries:

• Select conserved positions for sequence swaps that minimize structural disruption

  • Critical swap positions include conserved methionine residues (e.g., Ctr4 Met122 and Met227, and Ctr5 Met31 and Met134)

• Create systematic domain exchanges:

  • N-terminal domain (NTD) swaps

  • Transmembrane domain (TMD) swaps

  • C-terminal domain (CTD) swaps

• Validate chimeric constructs through:

  • Expression verification (Western blot)

  • Localization studies (fluorescence microscopy with GFP fusions)

  • Functional complementation in appropriate mutant strains

This approach revealed that functional chimeras typically contain the Ctr4 central domain (with the essential Met-X₃-Met motif) and the Ctr5 CTD (important for trafficking), highlighting the unique properties and contributions of each protein to the complex .

What methodologies are effective for studying the dynamics of Ctr5 cellular localization?

Several approaches provide insights into the dynamic localization of Ctr5:

• Time-course fluorescence microscopy with GFP-tagged Ctr5

  • Reveals temporal changes in localization during cellular processes

  • Can be combined with various cellular markers

• Photoactivatable or photoconvertible fluorescent protein fusions

  • Enable pulse-chase experiments to track protein movement

• FRAP (Fluorescence Recovery After Photobleaching)

  • Measures protein mobility and membrane dynamics

• Live-cell imaging during copper concentration shifts

  • Captures real-time responses to changing copper levels

• Stable integration vectors for consistent expression

  • Provides reliable expression for long-term studies

  • Overcomes issues with unstable genomic loci caused by repetitive regions

These approaches have revealed that Ctr5 undergoes dynamic relocalization during meiosis and in response to changing copper concentrations, providing insights into its regulation and function .

How does the Ctr4-Ctr5 system differ from other eukaryotic copper transport systems?

The Ctr4-Ctr5 system in Sch. pombe displays several unique characteristics compared to other eukaryotic copper transporters:

These differences highlight the evolutionary diversification of copper transport mechanisms while maintaining core functional elements like the Met-X₃-Met motif in at least one component of the transport system .

What experimental strategies can determine the copper transport kinetics of the Ctr4-Ctr5 complex?

Researchers can employ several approaches to characterize the kinetics of copper transport:

• Radioactive 64Cu uptake assays

  • Provides direct measurement of transport activity

  • Can determine Km and Vmax values for the complex

• Competition assays with other metals

  • Establishes transport specificity and potential inhibitors

• pH dependence studies

  • Reveals mechanistic details of the transport process

• Site-directed mutagenesis of key residues

  • Identifies amino acids critical for transport function

  • Combined with the above assays to establish structure-function relationships

• Copper-dependent enzyme activity assays

  • Indirect measurement of transport efficiency

  • Examples include SOD1 and copper amine oxidase (Cao1) activity assays

Such studies have shown that disruption of ctr4+ and ctr6+ results in altered SOD1 activity and decreased levels of CAO activity, particularly during early- and middle-phase meiosis under copper-limiting conditions .

What are promising approaches for resolving the three-dimensional structure of the Ctr4-Ctr5 complex?

Several complementary approaches hold promise:

• Cryo-electron microscopy (cryo-EM)

  • Particularly suitable for membrane protein complexes

  • May reveal the arrangement of the 2:1 Ctr4:Ctr5 heterotrimer

• X-ray crystallography

  • Requires successful crystallization of the purified complex

  • May provide high-resolution structural information

• Cross-linking mass spectrometry

  • Can identify interaction interfaces between Ctr4 and Ctr5

  • Useful for validating structural models

• Molecular dynamics simulations

  • Can model the dynamic behavior of the complex in a membrane environment

  • Requires some initial structural information

• Single-particle analysis

  • May reveal heterogeneity in complex assembly

  • Useful for capturing different conformational states

Resolving the three-dimensional structure would provide critical insights into the mechanism of copper transport and the unique interdependence of Ctr4 and Ctr5 .

How might the Ctr4-Ctr5 complex interact with other components of copper homeostasis machinery?

This complex research question can be addressed through:

• Proximity-based labeling approaches (BioID, APEX)

  • Identifies proteins in close proximity to Ctr4-Ctr5

  • May reveal novel interaction partners

• Co-immunoprecipitation followed by mass spectrometry

  • Detects stable protein-protein interactions

  • Can identify components of larger complexes

• Genetic interaction screens

  • Reveals functional relationships with other genes

  • May identify components acting in the same pathway

• Imaging-based colocalization studies

  • Determines spatial relationships with other copper homeostasis proteins

  • Can reveal dynamic interactions under different conditions

• Copper chaperone interaction studies

  • Determines how copper is transferred from transporters to chaperones

  • Critical for understanding the complete copper delivery pathway

These approaches could reveal connections between the Ctr4-Ctr5 complex and other components involved in copper homeostasis, providing a more comprehensive understanding of copper metabolism in fission yeast .