Analysis of copper toxicity by western blotting of secretory proteins translocated via the Sec61 channel.

As a trace element, copper plays a vital role in regulating cellular homeostasis by serving as a cofactor for various enzymes. Therefore, maintaining an adequate copper concentration inside the cell is crucial. Even moderate dysregulation of homeostasis can lead to cytotoxicity, protein aggregation, resulting in cell death. One of the significant cellular consequences in Saccharomyces cerevisiae is the inhibition of Sec61-mediated protein translocation in the secretory pathway, resulting in the accumulation of the cytosolic form of precursor secretory proteins, such as Gas1 and CPY, in the cytosol. The accumulation of unprocessed precursor proteins in the cytosol provides a method to read out translocon dysfunction. This chapter describes experimental approaches for analysing copper-induced translocation defects in yeast, with a primary focus on Western blot-based detection of precursor and mature secretory protein species. Given the high conservation of copper homeostasis and the secretory pathway across eukaryotes, these methods offer a robust framework for investigating the molecular mechanisms underlying copper toxicity and its impact on protein folding and maturation. This biochemical assay further enables the identification and characterization of chemical or genetic modulators that mitigate copper-induced translocation defects. Compounds that restore Sec61 function or improve ER targeting and maturation reduce the accumulation of precursor forms and promote the recovery of mature protein species. Thus, this approach provides a robust platform for assessing copper toxicity and for screening chemical agents that rescue ER translocation and protein maturation under metal stress conditions.

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