To resolve the signaling mechanisms that mediate the starvation-induced processes of Dictyostelium sporulation and encystation, we performed insertional mutagenesis on cells harboring an mRFP-tagged spore gene. We isolated a mutant in kinkyA (knkA), a gene without known function, which formed fruiting bodies with a kinked stalk and lacking viable spores. Immunoprecipitation of lysates of KnkA-YFP-transformed knkA- cells yielded a mammalian BCAS3 homolog as a KnkA interactor. bcas3- phenocopied knkA- and Bcas3 colocalized with KnkA to puncta. Bcas3 shares sequence similarity with proppins (beta-propellors that bind phosphoinositides). Mutation of 2 Bcas3 residues that are essential for PtdIns3P binding in proppins prevented Bcas3 binding to PtdIns3P as well as punctate Bcas3 and KnkA localization. KnkA puncta also colocalized with small but not large vesicles that contain the autophagy protein Atg8 and were contiguous with the endoplasmic reticulum. knkA- and bcas3- cells showed a pronounced decrease of RFP-GFP-Atg8 in neutral early autophagosomes, indicating that KnkA and Bcas3 are required for macroautophagy/autophagy. Knockouts in atg7, atg5 or atg9 substantiated this finding by showing similar sporulation defects as knkA- and bcas3-. Defective Dictyostelium sporulation is evidently a useful diagnostic tool for the discovery of novel autophagy genes.Abbreviations: Atg: Autophagy-related; BCAS3: BCAS3 microtubule associated cell migration factor; cAMP: 3',5'-cyclic adenosine monophosphate; ER: endoplasmic reticulum; GFP: green fluorescent protein; PAS: phagophore assembly site; PRKA/PKA: protein kinase cAMP-dependent; Proppin: beta-propellers that bind phosphoinositides; PtdIns3P: phosphatidylinositol 3-phosphate; REMI: restriction enzyme-mediated insertional mutagenesis; RFP: red fluorescent protein; RT-qPCR: reverse transcriptase - quantitative polymerase chain reaction; WIPI: WD repeat domain, phosphoinositide interacting; YFP: yellow fluorescent protein.

中文翻译：

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proppin Bcas3 及其相互作用物 KinkyA 定位于早期吞噬细胞并调节自噬。

为了解决介导饥饿诱导的网基网柄菌孢子形成和包囊形成过程的信号机制，我们对带有 mRFP 标记的孢子基因的细胞进行了插入诱变。我们在 kinkyA (knkA) 中分离出一个突变体，这是一个没有已知功能的基因，它形成具有扭结茎并且缺乏可行孢子的子实体。KnkA-YFP 转化的 knkA- 细胞裂解物的免疫沉淀产生哺乳动物 BCAS3 同源物作为 KnkA 相互作用物。bcas3- phenocopied knkA- 和 Bcas3 与 KnkA 共定位到斑点。Bcas3 与proppins（结合磷酸肌醇的β-螺旋桨）具有序列相似性。proppins 中 PtdIns3P 结合所必需的 2 个 Bcas3 残基的突变阻止了 Bcas3 与 PtdIns3P 的结合以及点状 Bcas3 和 KnkA 定位。KnkA 点也与包含自噬蛋白 Atg8 的小但不大的囊泡共定位，并与内质网相连。knkA- 和 bcas3- 细胞在中性早期自噬体中显示出 RFP-GFP-Atg8 的显着减少，表明巨自噬/自噬需要 KnkA 和 Bcas3。atg7、atg5 或 atg9 中的敲除通过显示与 knkA- 和 bcas3- 类似的孢子形成缺陷证实了这一发现。有缺陷的网柄菌孢子形成显然是发现新自噬基因的有用诊断工具。BCAS3：BCAS3微管相关细胞迁移因子；cAMP：3',5'-环磷酸腺苷；ER：内质网；GFP：绿色荧光蛋白；PAS：吞噬细胞组装位点；PRKA/PKA：蛋白激酶 cAMP 依赖性；支撑：结合磷酸肌醇的β螺旋桨；PtdIns3P：3-磷酸磷脂酰肌醇；REMI：限制性内切酶介导的插入诱变；RFP：红色荧光蛋白；RT-qPCR：逆转录酶-定量聚合酶链反应；WIPI：WD 重复结构域，磷酸肌醇相互作用；YFP：黄色荧光蛋白。