Figure S1.
Identification of a set of Rab GTPases is degraded via macroautophagy. (A) HEK293T cells transiently expressing HA-GFP-Rab GTPases were treated by DMSO or Torin1 for 4 h, and the lysosomal cleavage of GFP-Rab GTPases was analyzed by western blot. Note that Rab1, Rab2, Rab3, Rab4, Rab4L, and Rab5 in Fig. 2 B and Fig. S1 A are for repeated use. (B) HEK293 or HEK293 Atg7 KO cells transiently expressing GFP-Rab GTPases were treated by DMSO or Torin1 for 4 h, and the cleavage of GFP-Rab GTPases was analyzed by western blot. Note that Rab1, Rab2, and Rab3 in Fig. 2 D and Fig. S1 B are for repeated use. (C) U2OS cells transiently expressing mCherry-GFP-Rab GTPases or mCherry-GFP-LC3B were treated by Torin1 for 2 h, mCherry+GFP− puncta indicate lysosomal degradation of GFP-Rab GTPases or GFP-LC3B. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (D and E) The levels of endogenous p62 in HeLa WT or Atg9 KO HeLa were measured by western blot and quantified in B. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). **P < 0.01. (F and G) The levels of endogenous p62 in HeLa WT or FIP200 KO HeLa were measured by western blot and quantified in D. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (H and I) HeLa WT, Penta KO HeLa, and Atg7 KO HeLa cells transiently expressing GFP-Rab5, were treated by Torin1 or Torin1 and Bafilomycin A1 for 0, 2 or 4 h, and quantification of cleavage GFP is shown in I. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (J and K) HeLa WT, Penta KO HeLa, and Atg7 KO HeLa cells transiently expressing GFP-Rab8, were treated by Torin1 or Torin1 and Bafilomycin A1 for 0, 2, or 4 h, and quantification of cleavage GFP is shown in K. Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ***P < 0.001. (L and M) HeLa WT, Penta KO HeLa and Atg7 KO HeLa cells transiently expressing GFP-Rab9, were treated by Torin1 or Torin1 and Bafilomycin A1 for 0, 2 or 4 h, and Quantification of cleavage GFP is shown in M. Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ***P < 0.001. (N) HEK293T cells transiently expressing HA-GFP-Rab GTPases WT or HA-GFP-Rab GTPases mutant (Cys→Ser), Rab1(204, 205); Rab5(212, 213); Rab6(206, 208); Rab7(205, 207); Rab8(204); Rab9(200, 201); Rab10(199, 200); Rab11(212, 213); Rab13(200); Rab14(213, 215); Rab18 (199, 203); Rab19(215, 217); Rab21(221, 222); Rab24(200, 201); Rab27(219, 221); Rab28(218); Rab30(199, 200); Rab32(224, 225); Rab33(235, 237); Rab34(257, 258); Rab35(200, 201); Rab36(332, 333); Rab38(205, 208); Rab43(210, 212) were treated by Torin1 for 0, 2, or 4 h, and the cleavage of GFP-Rab GTPases was analyzed by western blot. Molecular weight measurements are in kD. Source data are available for this figure: SourceData FS1.

Identification of a set of Rab GTPases is degraded via macroautophagy. (A) HEK293T cells transiently expressing HA-GFP-Rab GTPases were treated by DMSO or Torin1 for 4 h, and the lysosomal cleavage of GFP-Rab GTPases was analyzed by western blot. Note that Rab1, Rab2, Rab3, Rab4, Rab4L, and Rab5 in Fig. 2 B and Fig. S1 A are for repeated use. (B) HEK293 or HEK293 Atg7 KO cells transiently expressing GFP-Rab GTPases were treated by DMSO or Torin1 for 4 h, and the cleavage of GFP-Rab GTPases was analyzed by western blot. Note that Rab1, Rab2, and Rab3 in Fig. 2 D and Fig. S1 B are for repeated use. (C) U2OS cells transiently expressing mCherry-GFP-Rab GTPases or mCherry-GFP-LC3B were treated by Torin1 for 2 h, mCherry+GFP− puncta indicate lysosomal degradation of GFP-Rab GTPases or GFP-LC3B. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (D and E) The levels of endogenous p62 in HeLa WT or Atg9 KO HeLa were measured by western blot and quantified in B. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). **P < 0.01. (F and G) The levels of endogenous p62 in HeLa WT or FIP200 KO HeLa were measured by western blot and quantified in D. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (H and I) HeLa WT, Penta KO HeLa, and Atg7 KO HeLa cells transiently expressing GFP-Rab5, were treated by Torin1 or Torin1 and Bafilomycin A1 for 0, 2 or 4 h, and quantification of cleavage GFP is shown in I. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (J and K) HeLa WT, Penta KO HeLa, and Atg7 KO HeLa cells transiently expressing GFP-Rab8, were treated by Torin1 or Torin1 and Bafilomycin A1 for 0, 2, or 4 h, and quantification of cleavage GFP is shown in K. Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ***P < 0.001. (L and M) HeLa WT, Penta KO HeLa and Atg7 KO HeLa cells transiently expressing GFP-Rab9, were treated by Torin1 or Torin1 and Bafilomycin A1 for 0, 2 or 4 h, and Quantification of cleavage GFP is shown in M. Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ***P < 0.001. (N) HEK293T cells transiently expressing HA-GFP-Rab GTPases WT or HA-GFP-Rab GTPases mutant (Cys→Ser), Rab1(204, 205); Rab5(212, 213); Rab6(206, 208); Rab7(205, 207); Rab8(204); Rab9(200, 201); Rab10(199, 200); Rab11(212, 213); Rab13(200); Rab14(213, 215); Rab18 (199, 203); Rab19(215, 217); Rab21(221, 222); Rab24(200, 201); Rab27(219, 221); Rab28(218); Rab30(199, 200); Rab32(224, 225); Rab33(235, 237); Rab34(257, 258); Rab35(200, 201); Rab36(332, 333); Rab38(205, 208); Rab43(210, 212) were treated by Torin1 for 0, 2, or 4 h, and the cleavage of GFP-Rab GTPases was analyzed by western blot. Molecular weight measurements are in kD. Source data are available for this figure: SourceData FS1.

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