Aspergillus oryzae can secrete large amounts of enzymes. However, the production of abundant secretory proteins triggers the unfolded protein response (UPR) in the endoplasmic reticulum (ER), and it is not clear how ER-associated protein degradation (ERAD) contributes to bulk protein production in A. oryzae. Here we identified AoCdc48, the sole A. oryzae ortholog of Saccharomyces cerevisiae AAA+ ATPase Cdc48, a component of the ERAD machinery. We found that AoCdc48 localizes in both nuclei and cytoplasm. Generation of an Aocdc48 conditional mutant showed that Aocdc48 repression leads to reduced cell growth and aberrant hyphal morphology. When Aocdc48-repressed cells were cultured on starch-containing plates, the α-amylase-encoding gene amyB was about 1.3-fold higher expressed. Indeed, a halo produced by secreted amylase was seen on potato starch-containing plates even when there was almost no growth under Aocdc48 repression. Fluorescence microscopy revealed that although AmyB seemed to be secreted, various organelle distributions were aberrant in Aocdc48-repressed cells. We found that D1 AAA domain is crucial for cell viability. Finally, we show that Aocdc48-overexpression also causes defects of cell growth, colonial morphology and conidial formation. Collectively, our results suggest that AoCdc48 is essential for growth and organelle distribution but dispensable for amylase secretion.

中文翻译：

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米曲霉中 ERAD 相关 AAA+ ATPase Cdc48 的表征和功能分析

米曲霉可以分泌大量的酶。然而，大量分泌蛋白的产生会触发内质网 (ER) 中的未折叠蛋白反应 (UPR)，目前尚不清楚 ER 相关蛋白降解 (ERAD) 如何促进米曲霉中的大量蛋白产生。在这里，我们确定了 AoCdc48，它是酿酒酵母 AAA+ ATPase Cdc48（ERAD 机制的一个组成部分）的唯一 A.oryzae 直向同源物。我们发现 AoCdc48 位于细胞核和细胞质中。Aocdc48 条件突变体的生成表明 Aocdc48 镇压导致细胞生长减少和菌丝形态异常。当 Aocdc48 抑制的细胞在含淀粉的平板上培养时，α-淀粉酶编码基因 amyB 的表达高出约 1.3 倍。的确，即使在 Aocdc48 抑制下几乎没有生长，在含马铃薯淀粉的平板上也能看到由分泌的淀粉酶产生的晕圈。荧光显微镜显示，虽然 AmyB 似乎是分泌的，但在 Aocdc48 抑制的细胞中，各种细胞器分布是异常的。我们发现 D1 AAA 域对细胞活力至关重要。最后，我们表明 Aocdc48 过表达也会导致细胞生长、菌落形态和分生孢子形成的缺陷。总的来说，我们的结果表明 AoCdc48 对生长和细胞器分布是必不可少的，但对淀粉酶分泌却不是必需的。我们发现 D1 AAA 域对细胞活力至关重要。最后，我们表明 Aocdc48 过表达也会导致细胞生长、菌落形态和分生孢子形成的缺陷。总的来说，我们的结果表明 AoCdc48 对生长和细胞器分布是必不可少的，但对淀粉酶分泌却不是必需的。我们发现 D1 AAA 域对细胞活力至关重要。最后，我们表明 Aocdc48 过表达也会导致细胞生长、菌落形态和分生孢子形成的缺陷。总的来说，我们的结果表明 AoCdc48 对生长和细胞器分布是必不可少的，但对淀粉酶分泌却不是必需的。