Fig 2: OXI inhibits autophagosome-lysosome fusion by repressing PRDX2/RAB7A axis in CRC cells. (A) RKO and HCT116 cells were treated with OXI (30 μM) or RAPA (100 nM) for 24 h, and then dyed with LysoTracker (75 nM) for 30 min. (B) Western blotting analysis of Myosin, SNAP29, Dynactin, Dynein HC and RAB7A treated with or without OXI (30 μM). (C) Western blotting analysis of RAB7A in HCT116 and RKO cells transfected with Vector or HA-PRDX2 and treated with OXI (30 μM). (D) Western blotting analysis of RAB7A protein level in HCT116 and RKO cells transfected with HA-RAB7A plasmids for 48 h. (E-F) HCT116 and RKO cells were transiently transfected with mRFP-GFP-LC3 and HA-RAB7A, then treated with OXI (30 μM). Scale bars, 10 μm. (G) Quantification of the ratio of yellow puncta (autophagosome) versus red puncta (autolysosome). (H) The expression level of RAB7A in xenografts was evaluated by IHC. Scale bar: 50 μm. All data are means ± SD.

Fig 3: Rab39A is required for autophagy depends on its GTP loading and C-terminal prenylation.a Rab39A binding with HOPS complex depends on its GTP loading, but not C-terminal prenylation. 293T cells were transfected with indicated plasmids for 24 h. Cells were lysed and immunoprecipitated with Flag M2 agarose and then Immunoblotted with indicated antibodies. SE, short exposure; LE, long exposure. b Activation of Rab39A enables HOPS complex subunits recruited onto autophagic vesicles. U2OS cells were transfected with indicated plasmids. After treated with 50 nM Torin 1 for 3 h, cells were fixed and immune stained with indicated antibody. White-framed squares show the zoomed area. Scale bar, 10 µm. c Quantification of results of (b). Each point represents an image used for colocalization analysis, n = 37, 33, 39, 37, containing 101, 103, 102, 105 cells respectively. Significance was determined by two-tailed T test. P value is listed. The box extends from the 25th to 75th percentiles, and the whiskers go down to the smallest value and up to the largest. d Rab39A regulates autophagy flux depending on its GTP loading activation and C-terminal prenylation. Cells were transfected with indicated plasmids respectively for 24 h. After treated with or without 50 nM Torin 1 for 3 h, cell lysate was immunoblotted with indicated antibodies. *, non-specific band; red arrows indicated endogenous Rab39A (lower) or EGFP-Rab39A (upper). e, f Quantification of results of (d) LC3II levels (e) or p62 levels (f) from three independent experiments. LC3II and p62 value are first divided by the corresponding Tubulin value of the sample to obtain the LC3II/Tubulin ratio and p62/Tubulin ratio, which are then normalized to lane 1 sample. Data are mean ± SEM. Significance is determined by two-tailed T test P value is listed. ns, not significant. g Rab2-HOPS-Rab39A promoting lipid mixing of proteoliposomes requires GTP loading activation. HOPS-2, VPS39/VPS11 subcomplex of HOPS complex; HOPS-4, VPS16/VPS18/VPS33A/VPS41 subcomplex of HOPS complex. T, proteoliposomes incorporated with t-SNAREs (STX17-SNAP29); V, proteoliposomes incorporated with v-SNARE (VAMP8); a.u., arbitrary units. h Quantitative results of (g) at around 2000 sec from three independent replicated experiments. a.u., arbitrary units. Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Fig 4: Reconstitution of Human HOPS complex with Rab2 and Rab7 in STX17-SNAP29-VAMP8 mediated lipid mixing in vitro.a, b VPS39-Flag or VPS41-Flag directly interacts with Myc-Rab2 or Myc-Rab7. Flag M2 agarose was blocked with BSA or conjugated with purified VPS39-Flag or VPS41-Flag from 293 S cells, then incubated with purified Myc-Rab2 (a) or Myc-Rab7 (b) from E. coli respectively, and then washed and eluted for immunoblotting by indicated antibodies. c Schematic drawing of lipid mixing assay with in vitro reconstituted proteoliposomes. d Futile reconstitution of Human HOPS complex with Rab2 and Rab7 in STX17-SNAP29-VAMP8 mediated lipid mixing. HOPS-2, VPS39/VPS11 subcomplex of HOPS complex; HOPS-4, VPS16/VPS18/VPS33A/ VPS41 subcomplex of HOPS complex; T, proteoliposomes incorporated with t-SNAREs (STX17-SNAP29); V, proteoliposomes incorporated with v-SNARE (VAMP8); a.u., arbitrary units. e Quantitative of results of (d) at around 2000 sec from three independent replicated experiments. a.u., arbitrary units. Data are presented as mean values ± SEM. f Endogenous Rab39A interacts with HOPS complex subunits, majorly VPS39 and VPS41, and STX17, but not interacting with VPS11, VPS16, VPS18, VPS33A. 293T cells were transfected with empty vector or tagged protein expression plasmids as indicated. 24 h after transfection, cells were lysed and immunoprecipitated with Flag M2 agarose (Left panel) or HA agarose (Right panel). Immunoblotting was performed with indicated antibodies. *, non-specific band. Black arrow indicates endogenous Rab39A band. g STX17 directly interacts with Rab39A, but not Rab39B. Glutathione Sepharose beads bound with GST, GST-Rab39A, or GST-Rab39B were incubated with purified STX17 for one hour, and then washed and boiled for immunoblotting. h Direct binding of GST-Rab39A or GST-Rab39B to VPS39-Flag or VPS41-Flag in vitro. Glutathione Sepharose beads bound with GST, GST-Rab39A, or GST-Rab39B were incubated with purified VPS39-Flag or VPS41-Flag for one hour, and then washed and boiled for immunoblotting. Source data are provided as a Source Data file.

Fig 5: Rab39A-HOPS-Rab2 bridges autophagic vesicles and enhances fusion in vitro.a Rab39A-HOPS-Rab2 promotes lipid mixing of proteoliposomes reconstituted with autophagic SNAREs. HOPS-2, VPS39/VPS11 subcomplex of HOPS complex; HOPS-4, VPS16/VPS18/VPS33A/VPS41 subcomplex of HOPS complex; HOPS-6, pre-incubated VPS39/VPS11 subcomplex and VPS16/VPS18/VPS33A/VPS41 subcomplex of HOPS complex. T, proteoliposomes incorporated with t-SNAREs (STX17-SNAP29); V, proteoliposomes incorporated with v-SNARE (VAMP8); a.u., arbitrary units. b Quantitative of results of (a) at around 2000 sec from three independent replicated experiments. a.u., arbitrary units. Data are presented as mean values ± SEM. c Schematic drawing of content mixing assay. d Rab39A-HOPS-Rab2 promotes content-mixing of proteoliposomes reconstituted with autophagic SNAREs. HOPS-2, two-subunit HOPS subcomplex containing VPS39/VPS11; HOPS-4, four-subunit HOPS subcomplex containing VPS16/VPS18/VPS33A/VPS41 T, proteoliposomes incorporated with t-SNAREs (STX17-SNAP29); V, proteoliposomes incorporated with v-SNARE (VAMP8); a.u., arbitrary units. e Quantitative of results of (d) at around 2000 sec from three independent replicated experiments. a.u., arbitrary units. Data are presented as mean values ± SEM. f Representative Cryo-EM images of mixed proteoliposomes incorporated with Rab2 or Rab39A separately (Ctrl); or proteoliposomes incorporated with Rab2 preincubated with HOPS-2 and proteoliposomes incorporated with Rab39A preincubated with HOPS-4 (with HOPS). Enlarged images were shown in the white-framed squares. Red arrows indicate tethered proteoliposomes. Scale bars for large images, 200 nm. Scale bars for enlarged images, 100 nm. g Percentage of tethered proteoliposomes per 1.69 μm² observed area as captured by electron microscopy (n = 20). Data are analyzed by two-sided T test and are presented as mean values ± SD. The box extends from the 25th to 75th percentiles, and the whiskers go down to the smallest value and up to the largest. h Schematic model illustrates how the HOPS complex bridges autophagosome and lysosomes by a “hook-up” way. GTP-loaded Rab39A is enriched on the lysosomes, and subsequently recruits the HOPS-4 subcomplex. On the other hand, autophagosomal localized GTP-Rab2 recruits the HOPS-2 subcomplex. HOPS-2 and HOPS-4 “hook-up” to form HOPS-6 to tether autophagosome and lysosome, which leads to promote autophagosome-lysosome fusion driven by STX17-SNAP29-VAMP8. HOPS-6, HOPS complex containing VPS11, VPS16, VPS18, VPS33A, VPS39 and VPS41; V39, VPS39; V41, VPS41. Source data are provided as a Source Data file.