Abstract
High protein secretion capacity in filamentous fungi requires an extremely efficient system for protein synthesis, folding and transport. When the folding capacity of the endoplasmic reticulum (ER) is exceeded, a pathway known as the unfolded protein response (UPR) is triggered, allowing cells to mitigate and cope with this stress. In yeast, this pathway relies on the transcription factor Hac1, which mediates the up-regulation of several genes required under these stressful conditions. In this work, we identified and characterized the ortholog of the yeast HAC1 gene in the filamentous fungus Neurospora crassa. We show that its mRNA undergoes an ER stress-dependent splicing reaction, which in N. crassa removes a 23 nt intron and leads to a change in the open reading frame. By disrupting the N. crassa hac-1 gene, we determined it to be crucial for activating UPR and for proper growth in the presence of ER stress-inducing chemical agents. Neurospora is naturally found growing on dead plant material, composed primarily by lignocellulose, and is a model organism for the study of plant cell wall deconstruction. Notably, we found that growth on cellulose, a substrate that requires secretion of numerous enzymes, imposes major demands on ER function and is dramatically impaired in the absence of hac-1, thus broadening the range of physiological functions of the UPR in filamentous fungi. Growth on hemicellulose however, another carbon source that necessitates the secretion of various enzymes for its deconstruction, is not impaired in the mutant nor is the amount of proteins secreted on this substrate, suggesting that secretion, as a whole, is unaltered in the absence of hac-1. The characterization of this signaling pathway in N. crassa will help in the study of plant cell wall deconstruction by fungi and its manipulation may result in important industrial biotechnological applications.
Highlights
The endoplasmic reticulum (ER) is crucial for the production of membrane and secreted proteins and its function is under tight control
A small element in the 3’ UTR of the S. cerevisiae hac1 mRNA, which has been shown to be important of its splicing in vivo [39], appears to be conserved in the identified N. crassa hac-1 gene
The lowest free energy form of hac-1 mRNA conforms to this structure, similar to its counterparts in different organisms, with the predicted cleavage sites located on the loops and surrounding the predicted 23 nt intron (Fig 1C)
Summary
The endoplasmic reticulum (ER) is crucial for the production of membrane and secreted proteins and its function is under tight control. ER resident transmembrane sensors trigger a conserved signaling pathway known as the unfolded protein response (UPR) (reviewed in [1, 2]) Activation of these sensors lead to a major transcriptional program aimed at increasing folding capacity in the ER and adjusting the secretory pathway [3], while mediating a decrease in ER protein load through selective mRNA degradation and translational repression [4,5,6,7], as well as a global reduction in protein synthesis [8]. These mechanisms, along with others [9], work together to revert ER stress and re-attain protein folding homeostasis in the ER [10]
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