Figure 7. TBC1D15 binds ATG8 family proteins.

(A) The indicated cells transiently expressing YFP-GABARAPL1 (green) and mCherry-Parkin (red) were treated with valinomycin for 3 hr. Scale bars, 10 μm. (B) YFP-TBC1D15 overexpressed in HEK293 cells was subjected to binding assays with GST-fused proteins (GABA, L1, and L2 represent GABARAP, GABARAPL1, and GABARAPL2, respectively). 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel). Coomassie brilliant blue (CBB) staining shows GST-fusion proteins in bound fractions (lower panel). (C) Binding assay carried out as in (B) with GST-GABARAPL1 WT (GST-WT) or its Y49A/L50A mutant (GST-YL). Immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel) are shown. (D) Cell extracts from HEK293 overexpressed HA-TBC1D15 and YFP, YFP-GABARAPL1 (YFP-WT), or its Y49AL50A mutant (YFP-YL) were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies. (E–H) The indicated YFP-tagged TBC1D15 full-length, truncated, or point-mutant protein or YFP-Fis1 overexpressed in HEK293 cells were subjected to binding assays with recombinant GST-GABARAPL1. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel). (I) Summary of binding abilities of truncated or point-mutated TBC1D15 constructs. –, +, ++, and +++ indicates binding of recombinant GST-GABARAPL1 to less than 1%, 1–5%, 5–10%, and over 10%, respectively of the total YFP-TBC1D15 fragment. Yellow boxes indicate YFP tags. (J) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (K) The YFP-LC3 morphology of cells in (J) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.

DOI: http://dx.doi.org/10.7554/eLife.01612.021

Figure 7—figure supplement 1. Schematic representation of potential LIR motifs of TBC1D15.

The amino acid position (Pos.) of the conserved aromatic residue in the potential LIR motifs in human TBC1D15 is indicated. Red, acidic residues; Blue, basic residues.

DOI: http://dx.doi.org/10.7554/eLife.01612.022

Figure 7—figure supplement 2. TBC1D17 has the ability to bind Atg8 homologues.

(A) Protein sequence comparison of the LIR motif in TBC1D15 with that of TBC1D17. Underlined sequence denotes consensus LIR motif. Red, acidic residues; Blue, basic residues. (B) Overexpressed YFP-TBC1D17 was subjected to binding assays with GST-fused Atg8 proteins (GABA, L1, and L2 represent GABARAP, GABARAPL1, and GABARAPL2, respectively). 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel) and coomassie brilliant blue (CBB) staining (lower panel). (C) Binding assays of YFP-TBC1D17 were carried out with GST-fused GABARAPL1 WT (GST-WT) or its Y49AL50A mutant (GST-YL). Immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel) are shown. (D) Extracts from cells overexpressing both HA-TBC1D17 and YFP, YFP-GABARAPL1 (YFP-L1), its Y49AL50A mutant (YL), YFP-LC3B or its F52A/L53A mutant (FL) were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies. (E) YFP-TBC1D17 WT and F261A mutant overexpressed in HEK293 cells were subjected to a binding assay with recombinant GST-LC3B. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel).

DOI: http://dx.doi.org/10.7554/eLife.01612.023