The Lectin Chaperone Calnexin Is Involved in the Endoplasmic Reticulum Stress Response by Regulating Ca2+ Homeostasis in Aspergillus nidulans

Abstract

The Ca²⁺-mediated signaling pathway is crucial for environmental adaptation in fungi. Here we show that calnexin, a molecular chaperone located in the endoplasmic reticulum (ER), plays an important role in regulating the cytosolic free calcium concentration ([Ca²⁺]_c) in Aspergillus nidulans Inactivation of calnexin (ClxA) in A. nidulans caused severe defects in hyphal growth and conidiation under ER stress caused by the ER stress-inducing agent dithiothreitol (DTT) or high temperature. Importantly, defects in the ΔclxA mutant were restored by the addition of extracellular calcium. Furthermore, the CchA/MidA complex (the high-affinity Ca²⁺ channels), calcineurin (calcium/calmodulin-dependent protein phosphatase), and PmrA (secretory pathway Ca²⁺ ATPase) were required for extracellular calcium-based restoration of the DTT/thermal stress sensitivity in the ΔclxA mutant. Interestingly, the ΔclxA mutant exhibited markedly reduced conidium formation and hyphal growth defects under the low-calcium condition, which is similar to defects caused by mutations in MidA/CchA. Moreover, the phenotypic defects were further exacerbated in the ΔclxA ΔmidA ΔcchA mutant, which suggested that ClxA and MidA/CchA are both required under the calcium-limiting condition. Using the calcium-sensitive photoprotein aequorin to monitor [Ca²⁺]_c in living cells, we found that ClxA and MidA/CchA complex synergistically coordinate transient increase in [Ca²⁺]_c in response to extracellular calcium. Moreover, ClxA, in particular its luminal domain, plays a role in mediating the transient [Ca²⁺]_c in response to DTT-induced ER stress in the absence of extracellular calcium, indicating ClxA may mediate calcium release from internal calcium stores. Our findings provide new insights into the role of calnexin in the regulation of calcium-mediated response in fungal ER stress adaptation.IMPORTANCE Calnexin is a well-known molecular chaperone conserved from yeast to humans. Although it contains calcium binding domains, little is known about the role of calnexin in Ca²⁺ regulation. In this study, we demonstrate that calnexin (ClxA) in the filamentous fungus Aspergillus nidulans, similar to the high-affinity calcium uptake system (HACS), is required for normal growth and conidiation under the calcium-limiting condition. The ClxA dysfunction decreases the transient cytosolic free calcium concentration ([Ca²⁺]_c) induced by a high extracellular calcium or DTT-induced ER stress. Our findings provide the direct evidence that calnexin plays important roles in regulating Ca²⁺ homeostasis in addition to its role as a molecular chaperone in fungi. These results provide new insights into the roles of calnexin and expand knowledge of fungal stress adaptation.

Keywords: Aspergillus nidulans; ER stress; calcium signaling.