Figure S4.
TBC1D9A is a Rab11a GAP and interacts with Arl8b via its N-terminal region encompassing the GRAM1 domain. (A) Yeast two-hybrid assay to detect the interaction between TBC1D9B (WT and RYQ →A mutant) and different forms of Arl8b. Co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His media to confirm viability and interactions, respectively. (B) Percentage of cells showing relocalization of GFP or GFP-TBC1D9B (WT and its mutants) to mitochondria with HA-tagged Arl8b (Q75L)-MitoID (n = 3; each dot represents a single experiment). The values are represented as the mean ± SEM. (C) Structural model of interactions between Arl8b and the N-terminal fragment of TBC1D9B (1–410 residues) was generated using the AlphaFold3 tool and visualized using Chimera software. The cyan chain denotes Arl8b, and the orange chain indicates TBC1D9B (1–410 residues). (D) Yeast two-hybrid assay to detect interaction of TBC1D9B (WT or indicated mutants) with Arl8b (Q75L). Co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His media to confirm viability and interactions, respectively. In the assay, LC3B was used as a positive control for binding to TBC1D9B. (E) ConSurf server tool was used to predict the evolutionary conservation score (represented by a color-coded scale) of Arl8b binding–defective mutants of TBC1D9B (highlighted in blue) predicted by the AlphaFold3 server. (F) Kinetic analysis of GTP hydrolysis activity of Rab11a in the presence of TBC1D9A (WT or RYQ→A) or TBC1D9B (WT or RYQ→A) was performed by measuring the release of free inorganic phosphate (Pi) using malachite green reagent. Absorbance was recorded at 650 nm, and plotted values were calculated by subtracting the signal of GDP-loaded samples from GTP-loaded samples. (G) Yeast two-hybrid assay was performed to check the interaction of Arl8b (Q75L) and LC3B with TBC1D9A (WT or E95A). The co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His media to confirm viability and interactions, respectively. (H) Endogenous immunoprecipitation was performed by incubating HEK293T cell lysates with an anti-Arl8 antibody, followed by immunoblotting with the indicated antibodies. Source data are available for this figure: SourceData FS4. Refer to the image caption for details. Panel A shows yeast two-hybrid assay images demonstrating interactions between TBC1D9B wild-type or mutant proteins and different forms of Arl8b on selective media. Panel B shows a bar graph quantifying the percentage of cells displaying mitochondrial relocalization of GFP or GFP-TBC1D9B constructs with Arl8b Q75L-MitoID. Panel C shows a structural model illustrating the interaction between Arl8b and the N-terminal region of TBC1D9B generated using AlphaFold-based prediction. Panel D shows yeast two-hybrid assay images assessing interactions between TBC1D9B wild-type or mutants and Arl8b Q75L, with LC3B as a positive control. Panel E shows a conservation analysis plot highlighting evolutionarily conserved residues of TBC1D9B involved in Arl8b binding. Panel F shows a line graph depicting Rab11a GTP hydrolysis kinetics in the presence of TBC1D9A or TBC1D9B wild-type or mutant proteins. Panel G shows yeast two-hybrid assay images evaluating interactions between Arl8b Q75L or LC3B and TBC1D9A wild-type or E95A mutant. Panel H shows immunoblot images of endogenous immunoprecipitation demonstrating interaction between Arl8b and TBC1D9A in HEK293T cells.

TBC1D9A is a Rab11a GAP and interacts with Arl8b via its N-terminal region encompassing the GRAM1 domain. (A) Yeast two-hybrid assay to detect the interaction between TBC1D9B (WT and RYQ →A mutant) and different forms of Arl8b. Co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His media to confirm viability and interactions, respectively. (B) Percentage of cells showing relocalization of GFP or GFP-TBC1D9B (WT and its mutants) to mitochondria with HA-tagged Arl8b (Q75L)-MitoID (n = 3; each dot represents a single experiment). The values are represented as the mean ± SEM. (C) Structural model of interactions between Arl8b and the N-terminal fragment of TBC1D9B (1–410 residues) was generated using the AlphaFold3 tool and visualized using Chimera software. The cyan chain denotes Arl8b, and the orange chain indicates TBC1D9B (1–410 residues). (D) Yeast two-hybrid assay to detect interaction of TBC1D9B (WT or indicated mutants) with Arl8b (Q75L). Co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His media to confirm viability and interactions, respectively. In the assay, LC3B was used as a positive control for binding to TBC1D9B. (E) ConSurf server tool was used to predict the evolutionary conservation score (represented by a color-coded scale) of Arl8b binding–defective mutants of TBC1D9B (highlighted in blue) predicted by the AlphaFold3 server. (F) Kinetic analysis of GTP hydrolysis activity of Rab11a in the presence of TBC1D9A (WT or RYQ→A) or TBC1D9B (WT or RYQ→A) was performed by measuring the release of free inorganic phosphate (Pi) using malachite green reagent. Absorbance was recorded at 650 nm, and plotted values were calculated by subtracting the signal of GDP-loaded samples from GTP-loaded samples. (G) Yeast two-hybrid assay was performed to check the interaction of Arl8b (Q75L) and LC3B with TBC1D9A (WT or E95A). The co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His media to confirm viability and interactions, respectively. (H) Endogenous immunoprecipitation was performed by incubating HEK293T cell lysates with an anti-Arl8 antibody, followed by immunoblotting with the indicated antibodies. Source data are available for this figure: SourceData FS4.

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