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.

FIG 1

Bioinformatic identification and subcellular location of ClxA. (A) Schematic representation of the conserved motifs of calnexin homologs in A. nidulans, S. pombe, and H. sapiens. The cutline indicates the position of the predicted cleavable signal peptide. TM, transmembrane domain. (B) Comparision of predicted high-affinity calcium binding motifs. The numbers denote the place on the original sequence of the first residue in the motif. (C) ClxA localizes in the ER as shown by using GFP-tagged fusion proteins. DAPI (4[prime],6-diamidino-2-phenylindole) was used to visualize nuclei. (D) Western blot analysis using an anti-GFP antibody detected a fusion protein of ClxA-GFP with a size of approximately 100 kDa.

FIG 2

The sensitivity to thermal/DTT stress in the ΔclxA mutant can be restored by extracellular calcium. (A) Colony morphology comparison for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ at 30 or 42°C for 2.5 days. (B) Quantitative total conidial production for the strains shown in panel A. (C) Colony morphology comparison for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ and/or 2 mM DTT at 30°C for 2.5 days. (D) Quantitative total conidial production for the strains shown in panel C. Error bars represent standard deviations from three replicates. Different lowercase letters on the bars of each group represent significant differences among strains (Tukey's multiple-comparison test, P < 0.05).

FIG 3

MidA and CchA are required for the extracellular calcium-based restoration of the DTT/thermal stress sensitivity in the ΔclxA mutant. (A) Colony morphology comparison for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ at 30 or 42°C for 2.5 days. (B) Quantitative total conidial production for the strains shown in panel A. (C) Colony morphology comparison for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ and/or 2 mM DTT at 30°C for 2.5 days. (D) Quantitative total conidial production for the strains shown in panel C. Error bars represent standard deviation from three replicates. Different lowercase letters on the bars of each group represent significant differences among strains (Tukey's multiple-comparison test, P < 0.05).

FIG 4

The DTT/thermal stress sensitivity in the ΔclxA mutant can be rescued by extracellular Ca²⁺ in a calcineurin-dependent way. (A and C) Quantitative total conidial production for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ at 30 or 42°C for 2.5 days. (B and D) Quantitative total conidial production for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ and/or 2 mM DTT at 30°C for 2.5 days. Different lowercase letters on the bars of each group represent significant differences among strains (Tukey's multiple-comparison test, P < 0.05).

FIG 5

Loss of pmrA abolishes extracellular calcium-based restoration of the DTT/thermal sensitivity in the ΔclxA mutant. (A) Colony morphology comparison for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ at 30 or 42°C for 2.5 days. (B) Quantitative total conidial production for the strains shown in panel A. (C) Colony morphology comparison for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ and/or 2 mM DTT at 30°C for 2.5 days. (D) Quantitative total conidial production for the strains shown in panel C. Error bars represent standard deviations from three replicates. Different lowercase letters on the bars of each group represent significant differences among strains (Tukey's multiple-comparison test, P < 0.05).

FIG 6

ClxA mediates transient [Ca²⁺]_c in response to extracellular calcium stimulus. (A) Colony morphology comparison for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ or 2 mM EGTA at 30°C for 2.5 days. (B) Quantitative total conidial production for the strains shown in panel A. (C) Real-time monitoring of the [Ca²⁺]_c of indicated strains following a stimulus with 100 mM CaCl₂. (D) The peak of transient [Ca²⁺]_c of the indicated strains after treatment with 100 mM CaCl₂. Error bars represent standard deviations from three replicates. Different lowercase letters on the bars of each group represent significant differences among strains (Tukey's multiple-comparison test, P < 0.05).

FIG 7

ClxA mediates transient [Ca²⁺]_c in response to ER stress. (A) The peak of transient [Ca²⁺]_c of the indicated strains after treatment with EGTA and DTT. (B) The peak of transient [Ca²⁺]_c of the indicated strains after treatment with 100 mM CaCl₂. Error bars represent standard deviations from three replicates. Different lowercase letters on the bars of each group represent significant differences among strains (Tukey's multiple comparison test, P < 0.05). (C) Colony morphology comparison for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ at 30 or 42°C for 2.5 days. (D) Colony morphology comparison for the indicated strains grown on solid MM in the presence or absence of 200 mM CaCl₂ and/or 2 mM DTT at 30°C for 2.5 days.

FIG 8

A working model of how ClxA function regulates [Ca²⁺]_c homeostasis in A. nidulans. ClxA coordinates with MidA/CchA to regulate [Ca²⁺]_c homeostasis in response to extracellular calcium stimulus. ClxA regulates [Ca²⁺]_c homeostasis in response to ER stress in the absence of extracellular calcium.