Skip to main content
. 2023 Apr 24;120(18):e2211501120. doi: 10.1073/pnas.2211501120

Fig. 3.

Fig. 3.

Disruption of the interaction between Vac8 and Vac17 markedly impairs vacuole inheritance. (A) Interaction sites of Vac8 in Interfaces I and II are important for vacuole inheritance. Vacuoles of yeast strains expressing wild-type or mutant Vac8 were cultured with FM 4-64 in YPD medium at 30 °C for 30 min. After removing free dye, cells were resuspended in fresh YPD medium and further grown at 30 °C for 3 h. FM 4-64 fluorescence on vacuoles was observed by fluorescence microscopy. Representative images from each cell type are shown (Right), and the bar graph shows the quantification of vacuole inheritance (Left). More than 100 cells per strain were examined in each experiment. Data represent the means ± SEM (error bar; n = 3). (Scale bar: 5 μm). (B) The interaction site of Vac17 in Interface II is important for vacuole inheritance. Experiments were performed as described in Fig. 3A. Yeast cells expressing wild-type or mutant Vac17 were used. Representative images from each cell type are shown (Right) and the graph shows quantification of vacuole inheritance (Left). More than 100 cells per strain were examined in each experiment. Data represent the means ± SEM (error bar; n = 3). (Scale bar: 5 μm). (C) The L336R/F339R mutation largely abolishes the interaction between Vac8 and Vac17. Yeast spheroplasts were detergent-solubilized, and detergent-insoluble material was removed by centrifugation. The resulting postcentrifugation supernatants were precleared by incubation with protein A Sepharose and treated with anti-myc antibodies or control mouse IgG. Protein A Sepharose was then added, and bound proteins were eluted with SDS sample buffer for Sodium Dodecyl Sulfate (SDS)-PolyAcrylamide Gel Electrophoresis (PAGE) analysis followed by immunoblotting using anti-myc and anti-Vac8 antibodies.