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PtdIns4P restriction by hydrolase SAC1 decides specific fusion of autophagosomes with lysosomes.

ABSTRACT: Biogenesis of autophagosomes is the early step of macroautophagy/autophagy and requires membrane acquisition mainly from ER-Golgi-sourced precursor vesicles. Matured autophagosomes fuse with lysosomes for final degradation. However, how this selective fusion is determined remains elusive. Here, we identified Sac1 by a high throughput screen in Saccharomyces cerevisiae to show it was critical for autophagosome-lysosome fusion through its PtdIns4P phosphatase activity. Sac1 deficiency caused a dramatic increase of PtdIns4P at early Golgi apparatus and abnormal incorporation of PtdIns4P into Atg9 vesicles and autophagosomes, which caused failure to recruit SNARE proteins for autophagosome fusion with vacuoles. Sac1 function in autophagy was highly conserved from yeast to mammalian cells. Our work thus suggested that correct upstream lipid incorporation was important for downstream fusion step of autophagy and that Sac1 played a critical and ancient role in this surveillance of lipid integration.Abbreviations: Ape1: aminopeptidase Ι; ATG: autophagy related; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; ERGIC: Golgi apparatus and ER-Golgi intermediate compartment; HOPS: homotypic fusion and protein sorting complex; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4K: phosphoinositide-4-kinase; PtdIns4P: phosphatidylinositol-4-phosphate; SD-N: nitrogen starvation medium; SNARE: soluble N-ethylamide-sensitive factor attachment protein receptor.

SUBMITTER: Zhang H

PROVIDER: S-EPMC8386628 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Glioblastoma multiforme (GBM) is the most common brain malignancy insensitive to radiotherapy (RT). Although macroautophagy/autophagy was reported to be a fundamental factor prolonging the survival of tumors under radiotherapeutic stress, the autophagic biomarkers coordinated to radioresistance of GBM are still lacking in clinical practice. Here we established radioresistant GBM cells and identified their protein profiles using tandem mass tag (TMT) quantitative proteomic analysis. It was found that SDC1 and TGM2 proteins were overexpressed in radioresistant GBM cells and tissues and they contributed to the poor prognosis of RT. Knocking down SDC1 and TGM2 inhibited the fusion of autophagosomes with lysosomes and thus enhanced the radiosensitivity of GBM cells. After irradiation, TGM2 bound with SDC1 and transported it from the cell membrane to lysosomes, and then bound to LC3 through its two LC3-interacting regions (LIRs), coordinating the encounter between autophagosomes and lysosomes, which should be a prerequisite for lysosomal EPG5 to recognize LC3 and subsequently stabilize the STX17-SNAP29-VAMP8 QabcR SNARE complex assembly. Moreover, when combined with RT, cystamine dihydrochloride (a TGM2 inhibitor) extended the lifespan of GBM-bearing mice. Overall, our findings demonstrated the EPG5 tethering mode with SDC1 and TGM2 during the fusion of autophagosomes with lysosomes, providing new insights into the molecular mechanism and therapeutic target underlying radioresistant GBM.Abbreviations: BafA1: bafilomycin A1; CQ: chloroquine; Cys-D: cystamine dihydrochloride; EPG5: ectopic P-granules 5 autophagy tethering factor; GBM: glioblastoma multiforme; GFP: green fluorescent protein; LAMP2: lysosomal associated membrane protein 2; LIRs: LC3-interacting regions; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NC: negative control; RFP: red fluorescent protein; RT: radiotherapy; SDC1: syndecan 1; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TGM2: transglutaminase 2; TMT: tandem mass tag; VAMP8: vesicle associated membrane protein 8; WT: wild type.

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PtdIns4P restriction by hydrolase SAC1 decides specific fusion of autophagosomes with lysosomes.

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