Figure 2.
Identification of a set of Rab GTPases subjected to autophagic degradation. (A) The collection of human Rab GTPases. If there are multiple isoforms for one Rab GTPase, only isoform A was chosen on the screen. (B and C) HEK293T cells transiently expressing GFP-Rab GTPases were treated with DMSO or Torin1 for 4 h. Free GFP was detected by western blot. Summary of Rab GTPases screened by GFP cleavage assay. Note that Rab1, Rab2, Rab3, Rab4, Rab4L, and Rab5 in Fig. 2 B and Fig. S1 A are for repeated use. (D and E) Atg7 KO HEK293 cells transiently expressing GFP-Rab GTPases were treated with DMSO or Torin1 for 4 h. Free GFP was detected by western blot. Summary of Rab GTPases screened by GFP cleavage assay. Note that Rab1, Rab2, and Rab3 in Fig. 2 D and Fig. S1 B are for repeated use. (F and G) U2OS cells transiently expressing mCherry-GFP-LC3B or mCherry-GFP-Rab GTPases were treated by DMSO or Torin1 for 4 h, mCherry+GFP− puncta indicated lysosomal degradation, mCherry+GFP− puncta were included for quantification (N = 20 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (H) HeLa and Atg9 KO HeLa cells were treated by Torin1 or Torin1 and Bafilomycin A1 for 0, 2, or 4 h, and the levels of endogenous Rab2, Rab8, Rab9, and p62 were measured by western blot. (I) HeLa and FIP200 KO HeLa cells were treated by Torin1 or Torin1and Bafilomycin A1 for 0, 2, or 4 h, and the levels of endogenous Rab2, Rab8, Rab9, and p62 were measured by western blot. (J and K) HeLa WT, Penta KO HeLa, and Atg7 KO HeLa cells transiently expressing GFP-Rab2, 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 < 0.001. (L) HEK293T cells transiently expressing GFP-Rab GTPases or the prenylation-deficient mutants, were treated by Torin1 for 0, 2, or 4 h, and the cleavage of GFP-Rab GTPases was analyzed by western blot. Spot size correlates to band intensity (in Fig. S2 N). Followed by quantification of the band intensity using ImageJ software. (M) HEK293T cells were treated with Torin1 or Torin1 and Bafilomycin A1 for 0, 2, or 4 h, the levels of endogenous Rab1, 5, 8, 9, 18, 21, 27, 34 were measured by western blot. (N) Lysosome purification using the lysoIP method. Immunoblotting for protein markers of various subcellular compartments in whole-cell lysates, purified lysosomes, or Rab GTPases. Lysates were prepared from cells expressing 3 × HA-tagged TMEM192, treated with Torin1, Torin1, and Bafilomycin A1 or Torin1+Bafilomycin A1+Proteinase K. The protein levels of Rab2, 5, 8, 21, VAMP8, CSTD, GM130, CLIMP63, Lamin B1, TOMM20, and actin were analyzed by western blot. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F2.

Identification of a set of Rab GTPases subjected to autophagic degradation. (A) The collection of human Rab GTPases. If there are multiple isoforms for one Rab GTPase, only isoform A was chosen on the screen. (B and C) HEK293T cells transiently expressing GFP-Rab GTPases were treated with DMSO or Torin1 for 4 h. Free GFP was detected by western blot. Summary of Rab GTPases screened by GFP cleavage assay. Note that Rab1, Rab2, Rab3, Rab4, Rab4L, and Rab5 in Fig. 2 B and Fig. S1 A are for repeated use. (D and E) Atg7 KO HEK293 cells transiently expressing GFP-Rab GTPases were treated with DMSO or Torin1 for 4 h. Free GFP was detected by western blot. Summary of Rab GTPases screened by GFP cleavage assay. Note that Rab1, Rab2, and Rab3 in Fig. 2 D and Fig. S1 B are for repeated use. (F and G) U2OS cells transiently expressing mCherry-GFP-LC3B or mCherry-GFP-Rab GTPases were treated by DMSO or Torin1 for 4 h, mCherry+GFP− puncta indicated lysosomal degradation, mCherry+GFP− puncta were included for quantification (N = 20 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (H) HeLa and Atg9 KO HeLa cells were treated by Torin1 or Torin1 and Bafilomycin A1 for 0, 2, or 4 h, and the levels of endogenous Rab2, Rab8, Rab9, and p62 were measured by western blot. (I) HeLa and FIP200 KO HeLa cells were treated by Torin1 or Torin1and Bafilomycin A1 for 0, 2, or 4 h, and the levels of endogenous Rab2, Rab8, Rab9, and p62 were measured by western blot. (J and K) HeLa WT, Penta KO HeLa, and Atg7 KO HeLa cells transiently expressing GFP-Rab2, 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 < 0.001. (L) HEK293T cells transiently expressing GFP-Rab GTPases or the prenylation-deficient mutants, were treated by Torin1 for 0, 2, or 4 h, and the cleavage of GFP-Rab GTPases was analyzed by western blot. Spot size correlates to band intensity (in Fig. S2 N). Followed by quantification of the band intensity using ImageJ software. (M) HEK293T cells were treated with Torin1 or Torin1 and Bafilomycin A1 for 0, 2, or 4 h, the levels of endogenous Rab1, 5, 8, 9, 18, 21, 27, 34 were measured by western blot. (N) Lysosome purification using the lysoIP method. Immunoblotting for protein markers of various subcellular compartments in whole-cell lysates, purified lysosomes, or Rab GTPases. Lysates were prepared from cells expressing 3 × HA-tagged TMEM192, treated with Torin1, Torin1, and Bafilomycin A1 or Torin1+Bafilomycin A1+Proteinase K. The protein levels of Rab2, 5, 8, 21, VAMP8, CSTD, GM130, CLIMP63, Lamin B1, TOMM20, and actin were analyzed by western blot. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F2.

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