Rationale:Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Radiotherapy has long been an important treatment for GBM. Despite recent advances in tumor radiotherapy, the prognosis of GBM remains poor due to radioresistance. Autophagy has been reported as a basic factor to prolong the survival of tumor under radiation stress, but the molecular mechanism of how autophagy contributes to GBM radioresistance was still lacking.Methods:We established radioresistant GBM cells and identified their protein profiles by Tandem mass tag (TMT) quantitative proteomic analysis, then chose the radioresistant genes based on the TMT analysis of GBM cells and differentially expressed genes (DEGs) analysis of GEO database. Colony formation, flow cytometry, qPCR, western blotting, mRFP-GFP-LC3, transmission electron microscopy, immunofluorescence, and co-IP assays were conducted to investigate the regulation mechanisms among these new-found molecules.Results:Syndecan 1 (SDC1) and Transglutaminase 2 (TGM2) were both overexpressed in the radioresistant GBM cells and tissues, contributing to the dismal prognosis of radiotherapy. Mechanically, after irradiation, SDC1 carried TGM2 from cell membrane into cytoplasm, and transported to lysosomes by binding to flotillin 1 (FLOT1), then TGM2 recognized the betaine homocysteine methyltransferase (BHMT) on autophagosomes to coordinate the encounter between autophagosomes and lysosomes.Conclusions:The SDC1-TGM2-FLOT1-BHMT copolymer, a novel member of the protein complexes involved in the fusion of lysosomes and autophagosomes, maintained the autophagic flux in the irradiated tumor cells and ultimately enhanced radioresistance of GBM, which provides new insights of the molecular mechanism and therapeutic targets of radioresistant GBM.