Transition metal ions are essential in all organisms, being cofactors for many proteins responsible for vital biological functions, also involved in the biology of reproduction, the nervous and immune systems, in carcinogenesis, cell death, tissue regeneration, and epigenetic modifications. However, transition metal ions can also catalyze cytotoxic reactions, producing active free radicals (ROS) when in excess. Therefore, all organisms require mechanisms to tightly regulate the levels of these metal ions. Many human genetic diseases (e.g., Menkes, Wilson’s, Parkinson’s, Alzheimer’s, myelodysplasias, hemochromatosis, etc.) and severe malnutrition cause aberrant Cu/Fe homeostasis with detrimental health problems (Arnal et al., 2013; Bandmann et al., 2015; El-Youssef, 2003; Gaggelli et al., 2006; González-Domínguez et al., 2014; Kaler et al., 2008). Copper is included among these essential transition metals, and once inside the cells, it is incorporated as a catalytic or structural cofactor into a variety of proteins. In eukaryotes, including Saccharomyces cerevisiae, one of the primary ways in which copper levels are regulated, is through changes in the expression of genes required for copper uptake, cellular distribution, storage, and export.
In S. cerevisiae, copper is transported in its cuprous form (Cu+) following reduction by the metallo-reductases (Fre1, Fre7) and by high affinity (Ctr1) and low affinity (Fet4, Smf1) transporters (Vest et al., 2019). Once in the cytosol, copper is assembled, among other proteins, into the superoxide dismutase Sod1 by the copper chaperone Ccs1 (Furukawa et al., 2004; Yuan et al., 1995). The Cu/Zn superoxide dismutase (Sod1) is a homodimeric free radical scavenging enzyme that catalyzes the conversion of O2− to H2O2 and O2 (Miao and St. Clair, 2009). More recent evidence indicates that Sod1 acts as a regulatory protein for diverse cellular processes such as regulation of respiration in response to glucose and oxygen levels via interaction with the casein kinase pathway, it plays an important role in various human diseases, and starvation stimulates its activity in order to prevent oxidative damage and enhance survival and finally, it acts as a nuclear transcription factor to control the general oxidative stress transcriptional response (Che et al., 2016; Chung, 2017; Glasauer et al., 2014; Reddi and Culotta, 2013; Tsang et al., 2014, 2018). Different yeast mutants with Sod1 deficiencies have shown amino-acid auxotrophies and reduced growth rate under aerobic conditions (Vest et al., 2019). Structurally, the Sod1 monomer consists of an eight-stranded beta-barrel that binds a copper ion and a zinc ion that play both structural and catalytic roles (Tainer et al., 1983). Sod1 interacts with Ccs1 as a heterodimer. The structure of Ccs1 contains three defined domains: 1) βαββαβ-folded Atx1-like domain, 2) central Sod1-like domain, and 3) carboxyterminal domain (Lamb et al., 1999). Domains 1 and 3, bind Cu(I), whereas domain 2 is crucial for the interaction and activation of Sod1 (Culotta et al., 1997; Lamb et al., 2000; Schmidt et al., 2000). Ccs1 chaperone is required for Sod1 maturation and enzymatic activity unless cells lacking Ccs1 are cultured with high levels of copper ions (Boyd et al., 2019; Fetherolf et al., 2017; Schmidt et al., 1999). Both Sod1 and Ccs1 are widely distributed throughout the cytosol, mitochondrial intermembrane space, and nucleus (Sturtz et al., 2001; Tsang et al., 2014; Wood and Thiele, 2009).
By tightly regulating the expression of the copper handling genes mentioned above, S. cerevisiae is able to balance copper levels despite the extracellular environment. Two transcription factors regulate copper gene expression. Ace1 is activated in response to high-copper conditions and activates the expression of genes that encode copper-binding metallothioneins and Sod1 superoxide dismutase (Labbé et al., 1997; Yamaguchi-Iwai et al., 1997). Conversely, in response to copper deficiency, Mac1 transcription factor, in synergy with other chromatin regulators, activates genes such as CTR1, FRE1, and FRE7 essential for copper reduction and import (Georgatsou and Alexandraki, 1999; Gkouskou et al., 2019; Gross et al., 2000; Jungmann et al., 1993; Labbé et al., 1997; Voutsina et al., 2019). The activity of these two transcription factors is coordinated so that their target genes are oppositely regulated in response to copper levels (Keller et al., 2005; Peña et al., 1998). In other organisms, such as Drosophila melanogaster and mammals, copper homeostasis is controlled at the transcriptional level by the zinc-responsive factor MTF-1 (Günther et al., 2012; Zhang et al., 2001).
Transcription factor Mac1 contains an amino-terminal DNA binding domain and a carboxyl-terminal activation domain and is localized to the nucleus in both copper-replete and copper-starvation conditions (Graden and Winge, 1997; Jungmann et al., 1993; Serpe et al., 1999). The amino-terminal (1–40) amino acids, include a copper-fist domain, which undergoes a conformational change on a copper-binding motif that allows DNA-binding. This domain contains a conserved, among fungal species, array of zinc-binding residues (Cys-X2-Cys-X8-Cys-X-His) (Turner et al., 1998). The carboxy-terminal activation domain contains two cysteine-rich, REPI and REPII copper-binding motifs (Brown et al., 2002; Jensen and Winge, 1998; Voutsina et al., 2001). Each one of these motifs binds four copper ions in a poly-copper cluster. In elevated copper levels, an intramolecular interaction between the DNA binding and transactivation domain results in the loss of both DNA binding and transactivation activity of Mac1 (Graden and Winge, 1997; Labbé et al., 1997). A point mutation, from histidine to glutamine (H279Q), in the REPI domain, leads to a constitutively active Mac1up protein that binds fewer copper ions per molecule than the wild-type protein (Graden and Winge, 1997; Jensen and Winge, 1998; Keller et al., 2000; Serpe et al., 1999). While this copper-induced allosteric switch explains how Mac1 activity is regulated by copper, the mechanism for copper translocation to the nucleus for binding to Mac1 remains unknown.
Previous studies suggested that activation of Mac1-regulated genes under copper-limiting conditions requires a functional Sod1 and that Mac1 activity can be affected by the DNA damaging agent MMS or by oxidative stress in a Sod1-dependent manner, suggesting that other environmental factors alter, and/or are critical to copper sensing (Dong et al., 2013; Tsang et al., 2014; Wood and Thiele, 2009). Many questions have been raised, regarding the mechanism by which Sod1 accomplishes Mac1 activation and whether additional proteins affect copper sensing and homeostasis.
In this study, based on a newly identified synthetic growth deficiency phenotype of the double mutant mac1Δ sod1Δ, we extended previous findings on the Sod1-Mac1 liaison, independently of prolonged copper deficiency or oxidative stress conditions. We present a weak, possibly transient physical interaction between Sod1 and Mac1 proteins, important for the specificity of the Sod1 enzymatic activity on Mac1 function. We showed that Sod1 modulates both DNA binding and transactivation functions of Mac1, while this relationship becomes Sod1-independent in the constitutively active mutant (copper-independent) Mac1up. Therefore, our findings point to the copper-related conformational alteration that the transcription factor undergoes due to the catalytic activity of Sod1. This is a housekeeping property of Mac1 protein regulation, under nutrient rich, physiological growth conditions.