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Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17.

ABSTRACT: Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome-lysosome fusion is retained to some extent even in STX17 knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited autophagosome-lysosome fusion partially in wild-type and completely in STX17 KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate autophagosome-lysosome fusion.

SUBMITTER: Matsui T

PROVIDER: S-EPMC6080929 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17.

Matsui Takahide T Jiang Peidu P Nakano Saori S Sakamaki Yuriko Y Yamamoto Hayashi H Mizushima Noboru N

The Journal of cell biology 20180522 8

Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome- ...[more]

PMID: 29789439

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Project description: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.

Project description:UnlabelledSyntaxin 17 is an autophagosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein required for the fusion of autophagosomes with lysosomes to form autolysosomes and thereby to deliver the enclosed contents for degradation. Hepatitis C virus (HCV) induces autophagy. In light of the observation that the number of viral particles formed by HCV-infected cells is much greater than the number of infectious viral particles finally released by HCV-infected cells, the regulation of fusion between autophagosomes and lysosomes might fulfill a key function controlling the number of released virions. HCV-replicating cells possess a decreased amount of syntaxin 17 due to impaired expression and increased turnover of syntaxin 17. Overexpression of syntaxin 17 in HCV-replicating cells diminishes the number of released infectious viral particles and decreases the amount of intracellular retained viral particles by favoring the formation of autolysosomes, in which HCV particles are degraded. Inhibition of lysosomal acidification by bafilomycin rescues the decreased release of virions from syntaxin 17-overexpressing cells, while induction of autophagy by rapamycin enforces the impairment of release under these conditions. Vice versa, inhibition of syntaxin 17 expression by specific small interfering RNAs results in an elevated amount of intracellular retained viral particles and facilitates the release of HCV virions by impairment of autophagosome-lysosome fusion. HCV genome replication, however, is not affected by modulation of syntaxin 17 expression. These data identify syntaxin 17 to be a novel factor controlling the release of HCV. This is achieved by regulation of autophagosome-lysosome fusion, which affects the equilibrium between the release of infectious viral particles and lysosomal degradation of intracellular retained viral particles.ImportanceHepatitis C virus (HCV) induces autophagy. Syntaxin 17 is an autophagosomal SNARE protein required for the fusion of autophagosomes with lysosomes. In HCV-infected cells, a major fraction of the de novo-synthesized viral particles is not released but is intracellularly degraded. In this context, the effect of HCV on the amount and distribution of syntaxin 17 and the relevance of syntaxin 17 for the viral life cycle were investigated. This study demonstrates that the amount of syntaxin 17 decreased in HCV-replicating cells. In addition, syntaxin 17 is identified to be a novel factor controlling the release of HCV, and the relevance of autophagosome-lysosome fusion as a regulator of the amount of released viral particles is revealed. Taken together, these findings indicate that syntaxin 17 is involved in the regulation of autophagosome-lysosome fusion and thereby affects the equilibrium between the release of infectious viral particles and the lysosomal degradation of intracellularly retained viral particles.

Dataset Information

Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17.

Publications

Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17.

Similar Datasets

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