Chalkophores.

Copper-binding metallophores, or chalkophores, play a role in microbial copper homeostasis that is analogous to that of siderophores in iron homeostasis. The best-studied chalkophores are members of the methanobactin (Mbn) family-ribosomally produced, posttranslationally modified natural products first identified as copper chelators responsible for copper uptake in methane-oxidizing bacteria. To date, Mbns have been characterized exclusively in those species, but there is genomic evidence for their production in a much wider range of bacteria. This review addresses the current state of knowledge regarding the function, biosynthesis, transport, and regulation of Mbns. While the roles of several proteins in these processes are supported by substantial genetic and biochemical evidence, key aspects of Mbn manufacture, handling, and regulation remain unclear. In addition, other natural products that have been proposed to mediate copper uptake as well as metallophores that have biologically relevant roles involving copper binding, but not copper uptake, are discussed.

Copper homeostasis and cuproptosis in cancer immunity and therapy.

Copper is an essential nutrient for maintaining enzyme activity and transcription factor function. Excess copper results in the aggregation of lipoylated dihydrolipoamide S-acetyltransferase (DLAT), which correlates to the mitochondrial tricarboxylic acid (TCA) cycle, resulting in proteotoxic stress and eliciting a novel cell death modality: cuproptosis. Cuproptosis exerts an indispensable role in cancer progression, which is considered a promising strategy for cancer therapy. Cancer immunotherapy has gained extensive attention owing to breakthroughs in immune checkpoint blockade; furthermore, cuproptosis is strongly connected to the modulation of antitumor immunity. Thus, a thorough recognition concerning the mechanisms involved in the modulation of copper metabolism and cuproptosis may facilitate improvement in cancer management. This review outlines the cellular and molecular mechanisms and characteristics of cuproptosis and the links of the novel regulated cell death modality with human cancers. We also review the current knowledge on the complex effects of cuproptosis on antitumor immunity and immune response. Furthermore, potential agents that elicit cuproptosis pathways are summarized. Lastly, we discuss the influence of cuproptosis induction on the tumor microenvironment as well as the challenges of adding cuproptosis regulators to therapeutic strategies beyond traditional therapy.

A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

The Copper Efflux Regulator (CueR).

The copper efflux regulator (CueR) is a classical member of the MerR family of metalloregulators and is common in gram-negative bacteria. Through its C-terminal effector-binding domain, CueR senses cytoplasmic copper ions to regulate the transcription of genes contributing to copper homeostasis, an essential process for survival of all cells. In this chapter, we review the regulatory roles of CueR in the model organism Escherichia coli and the mechanisms for CueR in copper binding, DNA recognition, and interplay with RNA polymerase in regulating transcription. In light of biochemical and structural analyses, we provide molecular details for how CueR represses transcription in the absence of copper ions, how copper ions mediate CueR conformational change to form holo CueR, and how CueR bends and twists promoter DNA to activate transcription. We also characterize the functional domains and key residues involved in these processes. Since CueR is a representative member of the MerR family, elucidating its regulatory mechanisms could help to understand the CueR-like regulators in other organisms and facilitate the understanding of other metalloregulators in the same family.

The physiological and pathophysiological roles of copper in the nervous system.

Copper is a critical trace element in biological systems due the vast number of essential enzymes that require the metal as a cofactor, including cytochrome c oxidase, superoxide dismutase and dopamine-ß-hydroxylase. Due its key role in oxidative metabolism, antioxidant defence and neurotransmitter synthesis, copper is particularly important for neuronal development and proper neuronal function. Moreover, increasing evidence suggests that copper also serves important functions in synaptic and network activity, the regulation of circadian rhythms, and arousal. However, it is important to note that because of copper's ability to redox cycle and generate reactive species, cellular levels of the metal must be tightly regulated to meet cellular needs while avoiding copper-induced oxidative stress. Therefore, it is essential that the intricate system of copper transporters, exporters, copper chaperones and copper trafficking proteins function properly and in coordinate fashion. Indeed, disorders of copper metabolism such as Menkes disease and Wilson disease, as well as diseases linked to dysfunction of copper-requiring enzymes, such as SOD1-linked amyotrophic lateral sclerosis, demonstrate the dramatic neurological consequences of altered copper homeostasis. In this review, we explore the physiological importance of copper in the nervous system as well as pathologies related to improper copper handling.

Cuproptosis and Cu: a new paradigm in cellular death and their role in non-cancerous diseases.

Cuproptosis, a newly characterized form of regulated cell death driven by copper accumulation, has emerged as a significant mechanism underlying various non-cancerous diseases. This review delves into the complex interplay between copper metabolism and the pathogenesis of conditions such as Wilson's disease (WD), neurodegenerative disorders, and cardiovascular pathologies. We examine the molecular mechanisms by which copper dysregulation induces cuproptosis, highlighting the pivotal roles of key copper transporters and enzymes. Additionally, we evaluate the therapeutic potential of copper chelation strategies, which have shown promise in experimental models by mitigating copper-induced cellular damage and restoring physiological homeostasis. Through a comprehensive synthesis of recent advancements and current knowledge, this review underscores the necessity of further research to translate these findings into clinical applications. The ultimate goal is to harness the therapeutic potential of targeting cuproptosis, thereby improving disease management and patient outcomes in non-cancerous conditions associated with copper dysregulation.

The Dynamic Plasticity of P. aeruginosa CueR Copper Transcription Factor upon Cofactor and DNA Binding.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E. coli CueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However, P. aeruginosa CueR, which shows high sequence similarity to E. coli CueR, has been less studied. Here, we applied room-temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics of P. aeruginosa CueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed that P. aeruginosa CueR is less dynamic than the E. coli CueR protein and exhibits much higher sensitivity to DNA binding as compared to its E. coli CueR homolog. Moreover, a difference in dynamical behavior was observed when P. aeruginosa CueR binds to the copZ2 DNA promoter sequence compared to the mexPQ-opmE promoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ-OpmE proteins in P. aeruginosa. Overall, such comparative measurements of protein-DNA complexes derived from different bacterial systems reveal insights about how structural and dynamical differences between two highly homologous proteins lead to quite different DNA sequence-recognition and mechanistic properties.

Adipocytes promote metastasis of breast cancer by attenuating the FOXO1 effects and regulating copper homeostasis.

BACKGROUND: Obesity and the forkhead box O1(FOXO1) affect the survival of breast cancer patients, but the underlying mechanism remains unclear. We aimed to investigate the role of FOXO1 in obesity-associated-breast cancer. METHODS: We screened 383 breast disease patients from the first affiliated hospital with Nanjing Medical University in 2020. We performed wound healing, transwell, matrigel assays to assess the metastatic ability of cancer cells. We adopted mRNAs sequencing to select the differentially expressed transcripts in breast cancer. We applied immunohistochemistry, western blot, tissue microarrays to assess the level of FOXO1 and epithelial-mesenchymal transition (EMT) pathways. We conducted bioinformatic analysis to investigate interactions between FOXO1 and miR-135b. We used fluorescence in situ hybridization, RT-qPCR to confirm the characteristics of circCNIH4. We conducted luciferase reporter assay, rescue experiments to investigate interactions between circCNIH4 and miR-135b. RESULTS: Obesity was positively correlated with the incidence and progression of breast cancer. Adipocytes enhanced the migration of breast cancer and attenuated the effects of FOXO1. MiR-135b was a binding gene of FOXO1 and was regulated by circCNIH4. CircCNIH4 exhibited antitumor activity in vitro and in vivo. CONCLUSION: Adipocytes might accelerate the progression of breast cancer by modulating FOXO1/miR-135b/ circCNIH4 /EMT axis and regulating copper homeostasis.

Regulation of copper uptake by the SWI/SNF chromatin remodeling complex in Candida albicans affects susceptibility to antifungal and oxidative stresses under hypoxia.

Candida albicans is a human colonizer and also an opportunistic yeast occupying different niches that are mostly hypoxic. While hypoxia is the prevalent condition within the host, the machinery that integrates oxygen status to tune the fitness of fungal pathogens remains poorly characterized. Here, we uncovered that Snf5, a subunit of the chromatin remodeling complex SWI/SNF, is required to tolerate antifungal stress particularly under hypoxia. RNA-seq profiling of snf5 mutant exposed to amphotericin B and fluconazole under hypoxic conditions uncovered a signature that is reminiscent of copper (Cu) starvation. We found that under hypoxic and Cu-starved environments, Snf5 is critical for preserving Cu homeostasis and the transcriptional modulation of the Cu regulon. Furthermore, snf5 exhibits elevated levels of reactive oxygen species and an increased sensitivity to oxidative stress principally under hypoxia. Supplementing growth medium with Cu or increasing gene dosage of the Cu transporter CTR1 alleviated snf5 growth defect and attenuated reactive oxygen species levels in response to antifungal challenge. Genetic interaction analysis suggests that Snf5 and the bona fide Cu homeostasis regulator Mac1 function in separate pathways. Together, our data underlined a unique role of SWI/SNF complex as a potent regulator of Cu metabolism and antifungal stress under hypoxia.

Exploring and clinical validation of prognostic significance and therapeutic implications of copper homeostasis-related gene dysregulation in acute myeloid leukemia.

The patterns and biological functions of copper homeostasis-related genes (CHRGs) in acute myeloid leukemia (AML) remain unclear. We explored the patterns and biological functions of CHRGs in AML. Using independent cohorts, including TCGA-GTEx, GSE114868, GSE37642, and clinical samples, we identified 826 common differentially expressed genes. Specifically, 12 cuproptosis-related genes (e.g., ATP7A, ATP7B) were upregulated, while 17 cuproplasia-associated genes (e.g., ATOX1, ATP7A) were downregulated in AML. We used LASSO-Cox, Kaplan-Meier, and Nomogram analyses to establish prognostic risk models, effectively stratifying patients with AML into high- and low-risk groups. Subgroup analysis revealed that high-risk patients exhibited poorer overall survival and involvement in fatty acid metabolism, apoptosis, and glycolysis. Immune infiltration analysis indicated differences in immune cell composition, with notable increases in B cells, cytotoxic T cells, and memory T cells in the low-risk group, and increased monocytes and neutrophils in the high-risk group. Single-cell sequencing analysis corroborated the expression characteristics of critical CHRGs, such as MAPK1 and ATOX1, associated with the function of T, B, and NK cells. Drug sensitivity analysis suggested potential therapeutic agents targeting copper homeostasis, including Bicalutamide and Sorafenib. PCR validation confirmed the differential expression of 4 cuproptosis-related genes (LIPT1, SLC31A1, GCSH, and PDHA1) and 9 cuproplasia-associated genes (ATOX1, CCS, CP, MAPK1, SOD1, COA6, PDK1, DBH, and PDE3B) in AML cell line. Importantly, these genes serve as potential biomarkers for patient stratification and treatment. In conclusion, we shed light on the expression patterns and biological functions of CHRGs in AML. The developed risk models provided prognostic implications for patient survival, offering valuable information on the regulatory characteristics of CHRGs and potential avenues for personalized treatment in AML.

Cuproptosis: unveiling a new frontier in cancer biology and therapeutics.

Copper plays vital roles in numerous cellular processes and its imbalance can lead to oxidative stress and dysfunction. Recent research has unveiled a unique form of copper-induced cell death, termed cuproptosis, which differs from known cell death mechanisms. This process involves the interaction of copper with lipoylated tricarboxylic acid cycle enzymes, causing protein aggregation and cell death. Recently, a growing number of studies have explored the link between cuproptosis and cancer development. This review comprehensively examines the systemic and cellular metabolism of copper, including tumor-related signaling pathways influenced by copper. It delves into the discovery and mechanisms of cuproptosis and its connection to various cancers. Additionally, the review suggests potential cancer treatments using copper ionophores that induce cuproptosis, in combination with small molecule drugs, for precision therapy in specific cancer types.

Deep learning enables the discovery of a novel cuproptosis-inducing molecule for the inhibition of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers in the world. The therapeutic outlook for HCC patients has significantly improved with the advent and development of systematic and targeted therapies such as sorafenib and lenvatinib; however, the rise of drug resistance and the high mortality rate necessitate the continuous discovery of effective targeting agents. To discover novel anti-HCC compounds, we first constructed a deep learning-based chemical representation model to screen more than 6 million compounds in the ZINC15 drug-like library. We successfully identified LGOd1 as a novel anticancer agent with a characteristic levoglucosenone (LGO) scaffold. The mechanistic studies revealed that LGOd1 treatment leads to HCC cell death by interfering with cellular copper homeostasis, which is similar to a recently reported copper-dependent cell death named cuproptosis. While the prototypical cuproptosis is brought on by copper ionophore-induced copper overload, mechanistic studies indicated that LGOd1 does not act as a copper ionophore, but most likely by interacting with the copper chaperone protein CCS, thus LGOd1 represents a potentially new class of compounds with unique cuproptosis-inducing property. In summary, our findings highlight the critical role of bioavailable copper in the regulation of cell death and represent a novel route of cuproptosis induction.

Copper(II) import and reduction are dependent on His-Met clusters in the extracellular amino terminus of human copper transporter-1.

Copper(I) is an essential metal for all life forms. Though Cu(II) is the most abundant and stable state, its reduction to Cu(I) via an unclear mechanism is prerequisite for its bioutilization. In eukaryotes, the copper transporter-1 (CTR1) is the primary high-affinity copper importer, although its mechanism and role in Cu(II) reduction remain uncharacterized. Here we show that extracellular amino-terminus of human CTR1 contains two methionine-histidine clusters and neighboring aspartates that distinctly bind Cu(I) and Cu(II) preceding its import. We determined that hCTR1 localizes at the basolateral membrane of polarized MDCK-II cells and that its endocytosis to Common-Recycling-Endosomes is regulated by reduction of Cu(II) to Cu(I) and subsequent Cu(I) coordination by the methionine cluster. We demonstrate the transient binding of both Cu(II) and Cu(I) during the reduction process is facilitated by aspartates that also act as another crucial determinant of hCTR1 endocytosis. Mutating the first Methionine cluster (7Met-Gly-Met9) and Asp13 abrogated copper uptake and endocytosis upon copper treatment. This phenotype could be reverted by treating the cells with reduced and nonreoxidizable Cu(I). We show that histidine clusters, on other hand, bind Cu(II) and are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity, as the second cluster is sufficient to preserve copper-induced CTR1 endocytosis upon complete deletion of the first cluster. We propose a novel and detailed mechanism by which the two His-Met-Asp residues of hCTR1 amino-terminus not only bind copper, but also maintain its reduced state, crucial for intracellular uptake.

Investigations of cellular copper metabolism in ovarian cancer cells using a ratiometric fluorescent copper dye.

Imbalances in metal homeostasis have been implicated in the progression and drug response of cancer cells. Understanding these changes will enable identification of new treatment regimes and precision medicine approaches to cancer treatment. In particular, there has been considerable interest in the interplay between copper homeostasis and response to platinum-based chemotherapeutic agents. Here, we have studied differences in the Cu uptake and distributions in the ovarian cancer cell line, A2780, and its cisplatin resistant form, A2780.CisR, by measuring total Cu content and the bioavailable Cu pool. Atomic absorption spectroscopy (AAS) revealed a lower total Cu uptake in A2780.CisR compared to A2780 cells. Conversely, live-cell confocal microscopy studies with the ratiometric Cu(I)-sensitive fluorescent dye, InCCu1, revealed higher relative cellular content of labile Cu in A2780.CisR cells compared with A2780 cells. These results demonstrate that Cu trafficking, homeostasis and speciation are different in the Pt-sensitive and resistant cells and may be associated with the predominance of different phenotypes for A2780 (epithelial) and A2780.CisR (mesenchymal) cells.

Principles to recover copper-conducting CTR proteins for the purpose of structural and functional studies.

Transition metals such as copper and zinc are essential elements required for the survival of most organisms, from bacteria to humans. Yet, elevated levels of these elements are highly toxic. The Copper TRansporter protein family (CTRs) represents the only identified copper uptake proteins in eukaryotes and hence serves as key components for the maintenance of appropriate levels of the metal. Moreover, CTRs have been proposed to serve as an entry point into cells of certain cancer drugs and to constitute attractive drug-targets for novel antifungals. Nevertheless, the structure, function, and regulation of the CTRs remain elusive, limiting valuable information also for applied sciences. To this end, here we report procedures to isolate a range of CTR members using Saccharomyces cerevisiae as a production host, focusing on three homologs, human CTR1, human CTR2, and Candida albicans CTR. Using forms C-terminally-linked to a protease cleavage sequence, Green Fluorescent Protein (GFP), and a His-tag, assessment of the localization, quantification and purification was facilitated. Cellular accumulation of the proteins was investigated via live-cell imaging. Detergents compatible with acceptable solubilization yields were identified and fluorescence-detection size-exclusion-chromatography (F-SEC) revealed preferred membrane extraction conditions for the targets. For purification purposes, the solubilized CTR members were subjected to affinity chromatography and SEC, reaching near homogeneity. The quality and quantity of the CTRs studied will permit downstream efforts to uncover imperative biophysical aspects of these proteins, paving the way for subsequent drug-discovery studies.

Copper removal capability and genomic insight into the lifestyle of copper mine inhabiting Micrococcus yunnanensis GKSM13.

Heavy metal pollution in mining areas is a serious environmental concern. The exploration of mine-inhabiting microbes, especially bacteria may use as an effective alternative for the remediation of mining hazards. A highly copper-tolerant strain GKSM13 was isolated from the soil of the Singhbhum copper mining area and characterized for significant copper (Cu) removal potential and tolerance to other heavy metals. The punctate, yellow-colored, coccoid strain GKSM13 was able to tolerate 500 mg L-1 Cu2+. Whole-genome sequencing identified strain GKSM13 as Micrococcus yunnanensis, which has a 2.44 Mb genome with 2176 protein-coding genes. The presence of putative Cu homeostasis genes and other heavy metal transporters/response regulators or transcription factors may responsible for multi-metal resistance. The maximum Cu2+ removal of 89.2% was achieved at a pH of 7.5, a temperature of 35.5 °C, and an initial Cu2+ ion concentration of 31.5 mg L-1. Alteration of the cell surface, deposition of Cu2+ in the bacterial cell, and the involvement of hydroxyl, carboxyl amide, and amine groups in Cu2+ removal were observed using microscopic and spectroscopic analysis. This study is the first to reveal a molecular-based approach for the multi-metal tolerance and copper homeostasis mechanism of M. yunnanensis GKSM13.

Structural Analyses of the Multicopper Site of CopG Support a Role as a Redox Enzyme.

Metal ions can be both essential components of cells as well as potential toxins if present in excess. Organisms utilize a variety of protein systems to maintain the concentration of metal ions within the appropriate range for cellular function, and to avoid concentrations where cellular damage can occur. In bacteria, numerous proteins contribute to copper homeostasis, including copper transporters, chelators, and redox enzymes. The genes that encode these proteins are often found in clusters, thus providing modular components that work together to achieve homeostasis. A better understanding of how these components function and cooperate to achieve metal ion resistance is needed, given the extensive use of metal ions, including copper, as broad-spectrum biocides in a variety of clinical and environmental settings. The copG gene is a common component of such copper resistance clusters, but its contribution to copper resistance is not well understood. In this review the available information about the CopG protein encoded by this gene is summarized. Comparison of the recent structure to diverse copper-containing metallochaperones, metalloenzymes, and electron transfer proteins suggests that CopG is a redox enzyme that uses multiple copper ions as active site redox cofactors to act on additional copper ion substrates. Mechanisms for both oxidase and reductase activity are proposed, and the biological advantages that these activities can contribute in conjunction with existing systems are described.

Interference between copper transport systems and platinum drugs.

Cisplatin, or cis-diamminedichloridoplatinum(II) cis-[PtCl2(NH3)2], is a platinum-based anticancer drug largely used for the treatment of various types of cancers, including testicular, ovarian and colorectal carcinomas, sarcomas, and lymphomas. Together with other platinum-based drugs, cisplatin triggers malignant cell death by binding to nuclear DNA, which appears to be the ultimate target. In addition to passive diffusion across the cell membrane, other transport systems, including endocytosis and some active or facilitated transport mechanisms, are currently proposed to play a pivotal role in the uptake of platinum-based drugs. In this review, an updated view of the current literature regarding the intracellular transport and processing of cisplatin will be presented, with special emphasis on the plasma membrane copper permease CTR1, the Cu-transporting ATPases, ATP7A and ATP7B, located in the trans-Golgi network, and the soluble copper chaperone ATOX1. Their role in eliciting cisplatin efficacy and their exploitation as pharmacological targets will be addressed.

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation.

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

The carboxy terminal transmembrane domain of SPL7 mediates interaction with RAN1 at the endoplasmic reticulum to regulate ethylene signalling in Arabidopsis.

In Arabidopsis, copper (Cu) transport to the ethylene receptor ETR1 mediated using RAN1, a Cu transporter located at the endoplasmic reticulum (ER), and Cu homeostasis mediated using SPL7, the key Cu-responsive transcription factor, are two deeply conserved vital processes. However, whether and how the two processes interact to regulate plant development remain elusive. We found that its C-terminal transmembrane domain (TMD) anchors SPL7 to the ER, resulting in dual compartmentalisation of the transcription factor. Immunoprecipitation coupled mass spectrometry, yeast-two-hybrid assay, luciferase complementation imaging and subcellular co-localisation analyses indicate that SPL7 interacts with RAN1 at the ER via the TMD. Genetic analysis revealed that the ethylene-induced triple response was significantly compromised in the spl7 mutant, a phenotype rescuable by RAN1 overexpression but not by SPL7 without the TMD. The genetic interaction was corroborated by molecular analysis showing that SPL7 modulates RAN1 abundance in a TMD-dependent manner. Moreover, SPL7 is feedback regulated by ethylene signalling via EIN3, which binds the SPL7 promoter and represses its transcription. These results demonstrate that ER-anchored SPL7 constitutes a cellular mechanism to regulate RAN1 in ethylene signalling and lay the foundation for investigating how Cu homeostasis conditions ethylene sensitivity in the developmental context.