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.
Keywords: Atg8 processing; Dictyostelium; Knka; breast carcinoma amplified sequence 3; inositol phospholipid binding; phagophore assembly.