Abstract
Secretory proteins are subjected to stringent quality control systems in the endoplasmic reticulum (ER) which include the targeting of misfolded proteins for proteasomal destruction via the ER‐associated degradation (ERAD) pathway. Since deletion of ERAD genes in the filamentous fungus Aspergillus niger had hardly any effect on growth, this study investigates whether autophagy might function as an alternative process to eliminate misfolded proteins from the ER. We generated A. niger double mutants by deleting genes essential for ERAD (derA) and autophagy (atg1 or atg8), and assessed their growth both under normal and ER stress conditions. Sensitivity toward ER stress was examined by treatment with dithiothreitol (DTT) and by expressing a mutant form of glucoamylase (mtGlaA::GFP) in which disulfide bond sites in GlaA were mutated. Misfolding of mtGlaA::GFP was confirmed, as mtGlaA::GFP accumulated in the ER. Expression of mtGlaA::GFP in ERAD and autophagy mutants resulted in a twofold higher accumulation in ΔderA and ΔderAΔatg1 strains compared to Δatg1 and wild type. As ΔderAΔatg1 mutants did not show increased sensitivity toward DTT, not even when mtGlaA::GFP was expressed, the results indicate that autophagy does not act as an alternative pathway in addition to ERAD for removing misfolded proteins from the ER in A. niger.
Highlights
Folding and post-translational modification of secretory and transmembrane proteins in eukaryotic cells takes place in the endoplasmic reticulum (ER), with the assistance of chaperones, foldases, and lectins
Compromising the ER-associated degradation (ERAD) pathway in the filamentous fungus A. niger only had a modest effect on growth (Carvalho et al 2011a), indicating that other mechanisms might be of importance to help the cells cope with ER stress
In order to investigate the possible involvement of the autophagy process in the removal of misfolded proteins from the ER, we deleted the autophagy-essential genes atg1 and atg8 in ERAD-defective A. niger ΔderA background strains expressing multiple copies of the heterologous GlaGus gene fusion
Summary
Folding and post-translational modification of secretory and transmembrane proteins in eukaryotic cells takes place in the endoplasmic reticulum (ER), with the assistance of chaperones, foldases, and lectins. Deletion of the der homolog derA in a heterologous protein overexpression strain of Aspergillus niger resulted in a sixfold increase in the intracellular accumulation of the protein accompanied by the induction of UPR target genes, but had no effect on growth and conidiation (Carvalho et al 2011a). Autophagy is a highly conserved, catabolic process responsible for the delivery of proteins, cytoplasmic components, and organelles to lytic compartments such as lysosomes in mammalian cells or vacuoles in plant and fungal cells It involves the sequestering of cytoplasmic contents in double membrane vesicles which subsequently fuse with the lysosome or vacuole, releasing their cargo.
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