Recombinant Arabidopsis thaliana Copper transporter 3 (COPT3)

Basic Research Questions

• What is the subcellular localization of COPT3 in Arabidopsis thaliana?

COPT3 is an intracellular copper transporter located in the endomembrane system, specifically in a compartment of the secretory pathway. Research has confirmed that COPT3 is not present in plastids or the plasma membrane. Subcellular fractionation experiments have shown that the COPT3 distribution pattern is more similar to ER protein markers than to mitochondrial or plasma membrane markers. Confocal microscopy using COPT3-GFP fusion proteins has verified an intracellular localization that excludes the plasma membrane and chloroplasts .

Methodologically, researchers have employed:

• COPT3-HA fusion proteins for immunodetection in cellular fractions

• Sucrose density gradient fractionation with specific antibodies against organelle markers

• Confocal microscopy of COPT3-GFP constructs in Arabidopsis protoplasts

• What is the tissue-specific expression pattern of COPT3?

COPT3 is expressed in multiple tissues but shows pronounced expression in reproductive organs and vascular tissue. Studies using RT-PCR analysis and promoter-GUS reporter constructs have revealed that:

• COPT3 expression is detectable in flowers, dried seeds, stems, and leaves

• Expression is hardly detectable in roots

• Strong expression occurs in pollen grains and anthers

• GUS staining is observed in stamen filaments when styles are elongating

• Clear expression appears in leaf vascular bundles

This expression profile suggests a role in copper transport in reproductive tissues and vascular systems .

• How is COPT3 expression regulated in response to copper levels?

COPT3 shows a complex regulation pattern that differs from other COPT family members:

• What is the basic structure and function of the COPT3 protein?

COPT3 (At5g59040) is a 151 amino acid protein with conserved features typical of COPT/CTR-type transport proteins:

• Contains three transmembrane domains (TMDs)

• Has an external amino terminus with a conserved Met residue

• Features a cytosolic carboxy terminus

• Contains conserved Mx₃M motif in TMD2 and Gx₃G motif in TMD3

Functionally, COPT3 acts as an intracellular copper transporter that:

• Exports copper from internal compartments

• Is involved in the interorgan reallocation of copper ions, particularly from vacuolar stores

• Participates in copper mobilization during certain developmental stages and in response to environmental conditions

Advanced Research Questions

• How does the TCP16 transcription factor regulate COPT3 expression at the molecular level?

TCP16 acts as a transcriptional repressor of COPT3, with complex temporal regulation:

• TCP16 directly binds to the COPT3 promoter through a specific CARE element (TTGAGCCCAT) as demonstrated by Electrophoretic Mobility Shift Assay (EMSA)

• The binding is specific, as shown by competition assays with unlabeled probes

• When TCP16 is overexpressed in plants, COPT3 expression is reduced to 25-15% of normal levels

• TCP16 expression is higher at 0h than at 12h under copper deficiency, creating an inverse relationship with COPT3 expression

• Under copper excess, TCP16 expression remains high throughout the day, coinciding with reduced COPT3 expression

• The TCP16 promoter contains an SPL7-responsive GTAC box, suggesting copper-responsive regulation

TCP16 thus contributes to the temporal regulation of internal copper mobilization, inhibiting copper remobilization at dawn through repression of COPT3 .

Methodologically, this interaction was studied using:

• EMSA with purified TCP16 protein and labeled DNA probes

• TCP16 overexpression lines with β-estradiol-inducible systems

• RT-qPCR to measure expression levels

• Temporal expression analysis under different copper conditions

• What are the phenotypic effects of COPT3 knockout or overexpression in Arabidopsis?

Altering COPT3 expression produces distinct phenotypes that reveal its physiological functions:

COPT3 Knockout Effects:

• Altered pollen morphology, consistent with TCP16 affecting pollen development

• Changes in copper content across different organs compared to wild-type plants

• Affects TCP16 expression, suggesting a feedback regulatory mechanism

COPT3 Overexpression Effects:

• Increased copper uptake and potential oxidative stress

• Phenotypes related to altered circadian rhythms

• When both COPT1 and COPT3 are overexpressed (COPT OE plants), plants show:

  • Altered iron homeostasis

  • Growth defects

  • Curly leaves phenotype similar to that observed when TCP16 is fused to a repressor domain

These findings indicate that COPT3 plays important roles in copper redistribution between organs, particularly from vegetative to reproductive structures, and that proper COPT3 expression is essential for normal development and metal homeostasis .

• What methodologies are most effective for studying recombinant COPT3 production and function?

Several effective approaches have been developed for studying recombinant COPT3:

For Recombinant Production:

• Expression in heterologous systems such as yeast complementation assays (using Saccharomyces cerevisiae copper transport mutants)

• Arabidopsis-based super-expression systems for preparative-scale production

• Tagged constructs (HA, GFP) for detection and localization studies

For Functional Analysis:

• Promoter-reporter fusions (COPT3p:GUS) for expression pattern analysis

• T-DNA insertion mutants and CRISPR/Cas9 for loss-of-function studies

• Inducible overexpression systems (such as β-estradiol-inducible promoters)

• Atomic absorption spectroscopy for copper content determination

• RT-qPCR for expression analysis under different conditions and timepoints

• Root elongation assays under varying copper conditions

• Electrophoretic Mobility Shift Assay (EMSA) for studying protein-DNA interactions

The Arabidopsis-based super-expression system has emerged as particularly valuable for recombinant protein production, offering advantages in terms of post-translational modifications, complex formation with endogenous interaction partners, and avoiding endotoxin contamination issues .

• How does COPT3 coordinate with other COPT family members in copper homeostasis?

COPT3 functions within a coordinated network of copper transporters that can be divided into two main subfamilies:

Plasma Membrane COPTs (pmCOPT):

• COPT1, COPT2, and COPT6

• Located at the plasma membrane

• Transcriptionally induced under copper deficiency

• Expression peaks at dawn (0h)

• Primarily responsible for copper uptake from the external environment

Internal Membrane COPTs (imCOPT):

• COPT3 and COPT5

• Located in internal membranes (endomembrane system, tonoplast)

• Not clearly induced by copper deficiency in the same pattern as pmCOPTs

• COPT3 expression peaks at dusk (12h)

• Responsible for mobilizing copper from internal stores

This subdivision distinguishes at least two copper sources in cells (external and internal), which are differentially mobilized based on the type of COPT activated. While COPT5 is well-characterized as a vacuolar copper exporter, COPT3 functions in a complementary compartment of the secretory pathway. Together, these transporters ensure proper copper distribution throughout the plant in response to changing environmental conditions and developmental stages .

• How is COPT3 function integrated with the plant's circadian rhythm?

COPT3 shows clear integration with circadian regulation:

• The COPT3 promoter contains regulatory elements conserved in light-regulated genes:

  • I-box (GATAA)

  • An element (CAANNNNATC) required for circadian expression

  • Evening Element (AAAATATCT) involved in circadian regulation

• According to the DIURNAL Database, COPT3 expression oscillates with a phase of around 24h under different circadian and diurnal conditions

• COPT3 expression peaks at 12h (end of light period) in a 12h light/12h dark cycle

• This pattern is opposite to plasma membrane COPT transporters, which peak at dawn

• Overexpression of COPT1 and COPT3 affects circadian rhythms regulation

This temporal organization may reflect the partitioning of copper uptake (dawn) versus internal redistribution (dusk) to optimize copper utilization throughout the day/night cycle and coordinate with other physiological processes .

Methodological approaches for studying this connection include:

• Temporal expression analysis under different photoperiod conditions

• Promoter element analysis

• Copper content measurements at different times of day

• Phenotypic characterization of circadian behaviors in COPT3 mutants

• What is the evolutionary conservation of COPT3 function across plant species?

The COPT family of transporters shows significant evolutionary conservation across eukaryotes:

• COPT transporters are part of the Ctr protein family conserved in eukaryotes for cellular copper acquisition

• The protein structure of Ctr/COPT members has been confirmed to contain three transmembrane segments that assemble as homotrimers or heterotrimers

• Copper is incorporated through a central pore in the assembled structure

While specific information about COPT3 conservation across plant species is limited in the provided search results, research shows that:

• The COPT family in Arabidopsis has high homology to other eukaryotic copper transporters (Ctr)

• Conserved features in COPT3 include three transmembrane domains, an external amino terminus containing a conserved Met residue, and the Mx₃M and Gx₃G motifs

• COPT3 is able to partially complement the respiratory and copper transport defect in a yeast ctr1Δctr3Δ mutant, demonstrating functional conservation across kingdoms

Understanding evolutionary conservation of COPT3 would benefit from:

• Comparative genomic analyses across plant species

• Functional complementation studies in other plants

• Structural comparisons of COPT3 homologs