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.

KEYWORDS:

• Autophagosome
• autophagy
• coincidence detection
• electrostatic interaction
• phosphoinositide
• PI4K2A

Acknowledgements

The authors thank Noboru Mizushima, Anne Spang, Gábor V Horváth for valuable discussions, and Erzsébet Fehér-Juhász for technical assistance.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2024.2322493

Additional information

Funding

The work was supported by the Magyar Tudományos Akadémia [LP2023-6]; Magyar Tudományos Akadémia [BO-00/761/21]; Magyar Tudományos Akadémia [BO-00/870/23]; Nemzeti Kutatási Fejlesztési és Innovációs Hivatal [K127961]; Nemzeti Kutatási Fejlesztési és Innovációs Hivatal [K146634]; Nemzeti Kutatási Fejlesztési és Innovációs Hivatal [PD128280]; Nemzeti Kutatási Fejlesztési és Innovációs Hivatal [UNKP-22-5-SZTE-575]; Nemzeti Kutatási Fejlesztési és Innovációs Hivatal [PD143268]; Nemzeti Kutatási Fejlesztési és Innovációs Hivatal [KKP129797]; Nemzeti Kutatási Fejlesztési és Innovációs Hivatal [UNKP-23-5-SZTE-681]; Nemzeti Kutatási Fejlesztési és Innovációs Hivatal [PD135587]; Tempus Közalapítvány [SHE-19314-004/2019].