A putative mitochondrial calcium uniporter in A. fumigatus contributes to mitochondrial Ca2+ homeostasis and stress responses

Abstract

Ca2+ uptake into mitochondria plays a central role in cell physiology by stimulating ATP production, shaping cytosolic Ca2+ transients and regulating cell survival or death. Although this system has been studied extensively in mammalian cells, the physiological implications of Ca2+ uptake into mitochondria in fungal cells are still unknown. In this study, a bi-directional best-hit BLASTP search revealed that the genome of Aspergillus fumigatus encodes a homolog of a putative mitochondrial Ca2+ uniporter (MCU) and a mitochondrial carrier protein AGC1/MICU1 homolog. Both putative homologs are mitochondrially localized and required for the response to azole and oxidative stress such that the loss of either McuA or AgcA results in reduced susceptibility to azole and oxidative stress, suggesting a role in environmental stress adaptation. Overexpressing mcuA restores the azole-resistance phenotype of the ΔagcA strain to wild-type levels, but not vice versa, indicating McuA plays a dominant role during these stress responses. Using a mitochondrially targeted version of the calcium-sensitive photoprotein aequorin, we found that only mcuA deletion leads to dysfunctional [Ca2+]mt and [Ca2+]c homeostasis, suggesting that McuA, but not AgcA, contributes to Ca2+ uptake into mitochondria. Further point-mutation experiments combined with extracellular Ca2+ chelator treatment verified that two predicted Ca2+-binding sites in McuA are required for Ca2+ uptake into mitochondria and stress responses through the regulation of [Ca2+]c homeostasis.