ABSTRACT

PROPPINs are conserved PtdIns3P-binding proteins required for autophagosome biogenesis that fold into a characteristic group of seven-bladed beta-propellers. Mutations in WDR45/WIPI4, a human member of this family, lead to BPAN, a rare form of neurodegeneration. We have generated mutants for the two PROPPIN proteins present in the model system Dictyostelium discoideum (Atg18 and Wdr45l) and characterized their function. Lack of Wdr45l greatly impairs autophagy, while Atg18 only causes subtle defects in the maturation of autolysosomes. The strong phenotype of the Wdr45l mutant is strikingly similar to that observed in Dictyostelium cells lacking Vmp1, an ER protein required for omegasome formation. Common phenotypes include impaired growth in axenic medium, lack of aggregation, and local enrichment of PtdIns3P as determined by the use of lipid reporters. In addition, Vmp1 and Wdr45l mutants show a chronically active response to ER stress. For both mutants, this altered PtdIns3P localization can be prevented by the additional mutation of the upstream regulator Atg1, which also leads to recovery of axenic growth and reduction of ER stress. We propose that, in addition to an autophagy defect, local autophagy-associated PtdIns3P accumulation might contribute to the pathogenesis of BPAN by disrupting ER homeostasis. The introduction of BPAN-associated mutations in Dictyostelium Wdr45l reveals the impact of pathogenic residues on the function and localization of the protein.

摘要

PROPPIN 是自噬体生物发生所需的保守 PtdIns3P 结合蛋白，可折叠成一组特征性的七叶 β 螺旋桨。该家族的人类成员 WDR45/WIPI4 的突变导致 BPAN，这是一种罕见的神经退行性疾病。我们已经为模型系统盘基网柄菌（Atg18 和 Wdr45l）中存在的两种 PROPPIN 蛋白生成了突变体，并表征了它们的功能。缺乏 Wdr45l 极大地损害了自噬，而 Atg18 仅导致自溶酶体成熟的细微缺陷。Wdr45l 突变体的强表型与在网柄菌中观察到的惊人相似缺乏 Vmp1 的细胞，这是一种形成 omegasome 所需的 ER 蛋白。常见的表型包括无菌培养基中的生长受损、缺乏聚集和 PtdIns3P 的局部富集，这由使用脂质报告基因确定。此外，Vmp1 和 Wdr45l 突变体显示出对 ER 应激的长期活跃反应。对于这两种突变体，这种改变的 PtdIns3P 定位可以通过上游调节因子 Atg1 的额外突变来防止，这也导致无菌生长的恢复和 ER 应激的减少。我们提出，除了自噬缺陷外，局部自噬相关的 PtdIns3P 积累可能通过破坏 ER 稳态来促进 BPAN 的发病机制。在网柄菌中引入 BPAN 相关突变Wdr45l 揭示了致病残基对蛋白质功能和定位的影响。