The proppin Bcas3 and its interactor KinkyA localize to the early phagophore and regulate autophagy

To resolve the signaling mechanisms that mediate the starvation-induced processes of <i>Dictyostelium</i> sporulation and encystation, we performed insertional mutagenesis on cells harboring an mRFP-tagged spore gene. We isolated a mutant in <i>kinkyA</i> (<i>knkA</i>), a gene without known function, which formed fruiting bodies with a kinked stalk and lacking viable spores. Immunoprecipitation of lysates of KnkA-YFP-transformed <i>knkA⁻</i> cells yielded a mammalian BCAS3 homolog as a KnkA interactor. <i>bcas3⁻</i> phenocopied <i>knkA⁻</i> 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. <i>knkA⁻</i> and <i>bcas3⁻</i> 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 <i>atg7, atg5</i> or <i>atg9</i> substantiated this finding by showing similar sporulation defects as <i>knkA⁻</i> and <i>bcas3⁻</i>. Defective <i>Dictyostelium</i> sporulation is evidently a useful diagnostic tool for the discovery of novel autophagy genes. <b>Abbreviations:</b> Atg: Autophagy-related; BCAS3: BCAS3 microtubule associated cell migration factor; cAMP: 3ʹ,5ʹ-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