Fig 2: TRIM22 interacts with GABARAPs and mediates their lysosomal localization. (A) HEK293T cells were transfected with MYC-TRIM22, and lysates were incubated with the indicated GST-tagged proteins and precipitated with glutathione sepharose 4B beads. Precipitates were then immunoblotted as shown. (B) Schematic domain structure of TRIM22 and its deletion mutants. (C–E) HEK293T cells were transfected as indicated, and lysates were incubated with the indicated GST-tagged proteins and precipitated with glutathione sepharose 4B beads. Precipitates were then immunoblotted as shown. (F) HeLa cells were transfected with HA-tagged LC3A, LC3B, LC3C, GABARAP, GABARAPL1, or GABARAPL2 along with MYC-TRIM22. The cells were immunostained for HA (green) and MYC (red). (G) Wild-type HeLa cells or TRIM22-KO1 cells were transfected as indicated, and the cells were immunostained for CD63 (green) and HA (red). Scale bars: 10 μm.

Fig 3: The R321K variant inhibits the role of TRIM22 in autophagosome-lysosome fusion. (A,B) mRFP-EGFP-LC3B was stably expressed in wild-type HeLa cells, TRIM22-KO1 cells, and TRIM22-KO1 cells expressing 3×FLAG-TRIM22-WT or R321K. (A) The cells with fluorescent LC3B puncta were analyzed. Puncta with mRFP+/EGFP+ indicate autophagosomes, and puncta with mRFP+/EGFP− indicate autolysosomes. Graphs show median with 95% confidence interval of more than 30 cells per group. (B) Cells were analyzed using flow cytometry. The intensities of mRFP and EGFP signals from more than 2,000 cells were analyzed, and the percentages of cells in corresponding gates are indicated. (C) HEK293T cells were transfected as indicated, and lysates were immunoprecipitated with antibodies against MYC. Precipitates were then immunoblotted as shown. (D) HEK293T cells were transfected as indicated, and lysates were incubated with GST-GABARAP proteins and precipitated with glutathione sepharose 4B beads. Precipitates were then immunoblotted as shown. (E,F) Wild-type HeLa cells, TRIM22-KO1 cells, and TRIM22-KO1 cells expressing 3×FLAG-TRIM22-WT or R321K were transfected with HA-PLEKHM1. (E) The cells were immunostained as indicated. Colocalization of HA-PLEKHM1 with GABARAP (left) or GABARAPL1 (right) was analyzed via calculation of Pearson’s correlation coefficient. Graphs show median with 95% confidence interval of more than 20 cells per group. (F) Cells were subjected to PLA with anti-HA antibodies along with antibodies against GABARAP (left) or GABARAPL1 (right). The number of PLA dots was quantified. Graphs show median with 95% confidence interval of more than 30 cells per group. *p < 0.05; **p < 0.01; *** p < 0.001; n.s., not significant. Scale bars: 10 μm.

Fig 4: Reduction in MSP300 causes polyUB accumulation marked with the autophagy intermediates ref(2)P and Atg8a. (A–L) Muscle RNAi knockdown of MSP300. Confocal imaging (maximum intensity z-projections) of fixed third-instar larval musculature. Scale bars: 20 µm. (A–F) Antibodies against polyUB (green, A,D) and ref(2)P (red, B,M) in wild-type control larva (Mhc>+, A–C) and the MSP300 knockdown mutant (Mhc>Trip-MSP300, D–F). C and F represent overlayed images of polyUB (green) and ref(2)P (red). (G–L) Antibodies against polyUB (green, G,J) and Atg8a (red, H,K) in wild-type control larva (Mhc>+, G–I) and the MSP300 knockdown mutant (Mhc>Trip-MSP300, J–L). I and L represent overlayed images of polyUB (green) and Atg8a. (M) Size distribution of polyUB-positive surfaces (in cubic microns) within the volume examined in A, D, G and H. Imaging data was quantified using Imaris v9.7.2. Quantitative data are presented as mean±s.e.m. with actual value of the mean shown. Individual animals are indicated as separate points (n=12). Size data were binned to <1 µm3, 1–5 µm3 and >5 µm3. Note the left y-axis describes the histogram for <1 µm3 volume whereas the right y-axis scales the 1–5 µm3 and >5 µm3 histograms. The P-values indicated are from a two-tailed unpaired Student's t-test analysis. (N) Colocalization of ref(2)P or Atg8a with polyUB in MSP300 knockdown larvae. The percentage of Imaris-generated surfaces of ref(2)P (filled red circles) or Atg8a (open red circles) that lies within surfaces generated by polyUB (similar to Manders M1 coefficient) are shown for size fractionated data (<1 µm3, 1–5 µm3 and >5 µm3). Quantitative data are presented as a mean±s.e.m. percentage. Individual animals are indicated as separate points (n=6). The P-values indicated are from a two-tailed unpaired Student's t-test analysis.

Fig 5: Atg8a overexpression, in the form of mCh–GFP–Atg8a, restores polyUB–Atg8a colocalization without affecting polyUB aggregate accumulation. (A–E) Confocal imaging (maximum intensity z-projections) of fixed third-instar of larval body wall muscle 6 expressing the autophagy marker Atg8a, tagged with both GFP and mCh in an MSP300 knockdown (Mhc>TRiP-MSP300) animal and immunolabelled with antibodies to polyUB and Atg8a. Scale bar: 5 µm. (A) mCherry (red), (B) GFP (green), (C) polyclonal antibodies against Atg8a (magenta) (D) monoclonal antibodies against polyubiquitin (cyan) and (E) merge of polyUB and Atg8a. (F) Size distribution of polyUB-positive surfaces (in cubic microns). Imaging data was quantified using Imaris v9.7.2. Quantitative data are presented as mean±s.e.m. with actual value of the mean shown. Individual animals are indicated as separate points (n=6). MSP300 knockdown larvae without Atg8a overexpression (Mhc>TRiP-MSP300, filled blue circles) and with Atg8a overexpression (Mhc>TRiP-MSP300, GFP-mCh-Atg8a, filled red circles) are shown. Size data were binned to <1 µm3, 1–5 µm3 and >5 µm3. Note the left y-axis describes the histograms for <1 µm3 volumes whereas the right y-axis scales the 1–5 µm3 and >5 µm3 histogram. The P-values indicated are from a two-tailed unpaired Student's t-test analysis. (G) Colocalization of Atg8a with polyUB in MSP300 knockdown larvae with or without Atg8a overexpression. The percentage of Imaris-generated Atg8a surfaces that lies within surfaces generated by polyUB (similar to Manders M1 coefficient) are shown for size fractionated data (<1 µm3, 1–5 µm3 and >5 µm3) without (filled red circles) or with (open red circles) Atg8a overexpression. Quantitative data are presented as a mean±s.e.m. percentage. Individual animals are indicated as separate points (n=6). Note that the data for animals without Atg8a overexpression were taken from Fig. 3. The P-values indicated are from a two-tailed unpaired Student's t-test analysis.