Figure 3.
Bro1ΔBOD sorting of NBD-PC into the vacuolar lumen is dependent on the Vps4–ESCRT machinery. (A) Domain cartoon of Bro1 (aa 1–844) with interacting factors annotated. (B) Sample micrographs of WT (SEY6210), bro1Δ (GOY65), and bro1ΔBOD (bro1Δ::TEF1p- bro1ΔBOD; CTY2) cells costained with NBD-PC and FM4-64, revealing endpoints of the observed phenotypes. Scale bars = 5 µm. (C) NBD-PC– and FM4-64–stained WT (SEY6210), TEF1p-BRO1 (CTY1), vps4Δ (MBY3), bro1Δ (GOY65), TEF1p- bro1ΔBOD (CTY2), TEF1p-bro1V (CTY4), bro1ΔBOD vps4Δ (CTY5), bro1ΔBOD vps27Δ (CTY29), bro1ΔBOD vps37Δ (CTY21), bro1ΔBOD vps22Δ (CTY24), bro1ΔBOD snf7Δ (CTY12), bro1ΔBOD vps4Δ(CTY5), and bro1ΔBOD doa4Δ(CTY13) cells were analyzed by live-cell fluorescence microscopy and quantified for the frequency of cells able to support NBD-PC trafficking to the vacuolar lumen. Data are represented as mean ± SEM. Asterisks indicate statistically significant differences (P < 0.006), and number signs indicate statistically significant difference compared with bro1Δ (P < 0.0001). Percentage of cells with WT sorting signal was quantified and calculated from at least 94 cells from four independent experiments performed on four different days. (D and E)bro1V (CTY4) were analyzed by ET. 3D reconstructions of the tomogram are shown in D, and ILVs per MVB are quantified in E. 20 MVBs from WT (SEY6210), 32 MVBs from bro1Δ (GOY65), 64 MVBs from bro1ΔBOD (CTY2) and 72 MVBs from bro1V (CTY4) were quantified. Data are represented as mean ± SEM. The limiting membrane of normal-like MVBs are labeled yellow, while the limiting membrane of tubular/aberrant MVBs are shown in different colors. ILVs are highlighted in red. A minimum of 13 MVBs from at least 10 cells were quantified. Scale bar = 100 nm. Asterisks indicate statistically significant differences comapred to bro1 (P < 0.0001). (F) WT (SEY6210), bro1Δ (GOY65), or bro1V (bro1Δ::TEF1p-bro1V; CTY4) cells were transformed with the GFP-CPS plasmid to assess MVB sorting using live-cell fluorescence microscopy. The percentage of cells with WT sorting signal was quantified and calculated from at least 156 cells from three independent experiments performed on three different days from two different transformations. White dashed lines indicate cell boundaries. Scale bars = 5 µm.

Bro1ΔBOD sorting of NBD-PC into the vacuolar lumen is dependent on the Vps4–ESCRT machinery. (A) Domain cartoon of Bro1 (aa 1–844) with interacting factors annotated. (B) Sample micrographs of WT (SEY6210), bro1Δ (GOY65), and bro1ΔBOD (bro1Δ::TEF1p- bro1ΔBOD; CTY2) cells costained with NBD-PC and FM4-64, revealing endpoints of the observed phenotypes. Scale bars = 5 µm. (C) NBD-PC– and FM4-64–stained WT (SEY6210), TEF1p-BRO1 (CTY1), vps4Δ (MBY3), bro1Δ (GOY65), TEF1p- bro1ΔBOD (CTY2), TEF1p-bro1V (CTY4), bro1ΔBOD vps4Δ (CTY5), bro1ΔBOD vps27Δ (CTY29), bro1ΔBOD vps37Δ (CTY21), bro1ΔBOD vps22Δ (CTY24), bro1ΔBOD snf7Δ (CTY12), bro1ΔBOD vps4Δ(CTY5), and bro1ΔBOD doa4Δ(CTY13) cells were analyzed by live-cell fluorescence microscopy and quantified for the frequency of cells able to support NBD-PC trafficking to the vacuolar lumen. Data are represented as mean ± SEM. Asterisks indicate statistically significant differences (P < 0.006), and number signs indicate statistically significant difference compared with bro1Δ (P < 0.0001). Percentage of cells with WT sorting signal was quantified and calculated from at least 94 cells from four independent experiments performed on four different days. (D and E)bro1V (CTY4) were analyzed by ET. 3D reconstructions of the tomogram are shown in D, and ILVs per MVB are quantified in E. 20 MVBs from WT (SEY6210), 32 MVBs from bro1Δ (GOY65), 64 MVBs from bro1ΔBOD (CTY2) and 72 MVBs from bro1V (CTY4) were quantified. Data are represented as mean ± SEM. The limiting membrane of normal-like MVBs are labeled yellow, while the limiting membrane of tubular/aberrant MVBs are shown in different colors. ILVs are highlighted in red. A minimum of 13 MVBs from at least 10 cells were quantified. Scale bar = 100 nm. Asterisks indicate statistically significant differences comapred to bro1 (P < 0.0001). (F) WT (SEY6210), bro1Δ (GOY65), or bro1V (bro1Δ::TEF1p-bro1V; CTY4) cells were transformed with the GFP-CPS plasmid to assess MVB sorting using live-cell fluorescence microscopy. The percentage of cells with WT sorting signal was quantified and calculated from at least 156 cells from three independent experiments performed on three different days from two different transformations. White dashed lines indicate cell boundaries. Scale bars = 5 µm.

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