Department Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
Appl Microbiol Biotechnol. 2011 Jan;89(2):357-73. doi: 10.1007/s00253-010-2916-5. Epub 2010 Oct 5.
Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase-glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation.
内质网相关降解（ERAD）是一种保守的机制，通过将错误折叠的蛋白质靶向到蛋白酶体进行降解，从而从内质网中去除这些蛋白质。为了评估 ERAD 在丝状真菌中的作用，我们研究了丝状真菌黑曲霉中破坏假定的 ERAD 成分的后果。在黑曲霉中缺失 derA、doaA、hrdC、mifA 或 mnsA 可产生存活的菌株，与亲本菌株相比，除了 doaA 之外，没有观察到明显的生长表型。在含有单拷贝或多拷贝葡糖淀粉酶-葡糖醛酸酶（GlaGus）基因融合的黑曲霉菌株中，也对基因缺失突变体进行了构建。未折叠蛋白反应（UPR）靶基因（bipA 和 pdiA）的诱导依赖于异源基因和 ERAD 基因的拷贝数。在含有多个 GlaGus 基因拷贝的 ERAD 突变体中，观察到 UPR 靶基因的最高诱导。Western blot 分析表明，在多拷贝 GlaGus 过表达菌株中缺失 derA 基因导致细胞内检测到的 GlaGus 蛋白增加了 6 倍。我们的结果表明，破坏 ERAD 途径的某些成分与异源蛋白的高水平表达相结合，导致细胞内蛋白质水平升高，表明蛋白质降解延迟。
