Abstract
Copper (Cu) chaperones, of which yeast ATX1 is a prototype, are small proteins with a Cu(I) binding MxCxxC motif and are responsible for directing intracellular Cu toward specific client protein targets that use Cu as a cofactor. The Chlamydomonas reinhardtii ATX1 (CrATX1) was identified by its high sequence similarity with yeast ATX1. Like the yeast homologue, CrATX1 accumulates in iron-deficient cells (but is not impacted by other metal-deficiencies). N- and C-terminally YFP-ATX1 fusion proteins are distributed in the cytoplasm. Reverse genetic analysis using artificial microRNA (amiRNA) to generate lines with reduced CrATX1 abundance and CRISPR/Cpf1 to generate atx1 knockout lines validated a function for ATX1 in iron-poor cells, again reminiscent of yeast ATX1, most likely because of an impact on metalation of the multicopper oxidase FOX1, which is an important component in high-affinity iron uptake. We further identify other candidate ATX1 targets owing to reduced growth of atx1 mutant lines on guanine as a sole nitrogen source, which we attribute to loss of function of UOX1, encoding a urate oxidase, a cupro-enzyme involved in guanine assimilation. An impact of ATX1 on Cu distribution in atx1 mutants is strikingly evident by a reduced amount of intracellular Cu in all conditions probed in this work.