PtdIns4P is required for the autophagosomal recruitment of STX17 (syntaxin 17) to promote lysosomal fusion.

ABSTRACT

The autophagosomal SNARE STX17 (syntaxin 17) promotes lysosomal fusion and degradation, but its autophagosomal recruitment is incompletely understood. Notably, PtdIns4P is generated on autophagosomes and promotes fusion through an unknown mechanism. Here we show that soluble recombinant STX17 is spontaneously recruited to negatively charged liposomes and adding PtdIns4P to liposomes containing neutral lipids is sufficient for its recruitment. Consistently, STX17 colocalizes with PtdIns4P-positive autophagosomes in cells, and specific inhibition of PtdIns4P synthesis on autophagosomes prevents its loading. Molecular dynamics simulations indicate that C-terminal positively charged amino acids establish contact with membrane bilayers containing negatively charged PtdIns4P. Accordingly, Ala substitution of Lys and Arg residues in the C terminus of STX17 abolishes membrane binding and impairs its autophagosomal recruitment. Finally, only wild type but not Ala substituted STX17 expression rescues the autophagosome-lysosome fusion defect of STX17 loss-of-function cells. We thus identify a key step of autophagosome maturation that promotes lysosomal fusion.Abbreviations Cardiolipin 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol; DMSO dimethyl sulfoxide; GST glutathione S-transferase; GUV giant unilamellar vesicles; LAMP1 lysosomal associated membrane protein 1; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; PA 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PC/POPC 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; PG 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); PI L-α-phosphatidylinositol; PI4K2A phosphatidylinositol 4-kinase type 2 alpha; PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3; POPE/PE 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine; PtdIns(3,5)P2 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-3',5'-bisphosphate); PtdIns3P 1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate); PtdIns4P 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-4'-phosphate); SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STX17 syntaxin 17.