bro1ΔBOD-supported ILV formation requires the Vps4–ESCRT machinery. This figure complements Fig. 3. (A) Lysates generated from WT (SEY6210), vps4Δ (MBY3), bro1Δ (GOY65), TEF1p-BRO1 (CTY1), bro1ΔBOD (370–844; bro1Δ::TEF1p-bro1ΔBOD; CTY2), bro1ΔBODΔUBD (388–844; bro1Δ::TEF1p-bro1ΔBODΔUBD; CTY3), TEF1p-bro1V (CTY4), and bro1ΔBOD vps4Δ (CTY5) cells were analyzed by immunoblotting using antibodies against Bro1, Vps4, and Pgk1. Numbers below the Bro1 blot indicate expression levels normalized to BRO1 expression. (B) WT (SEY6210), bro1ΔBOD (370–844; CTY2), bro1ΔBODΔUBD (388–844; CTY3), bro1ΔBOD hse1Δ (CTY30), bro1ΔBOD vps27Δ (CTY29), bro1ΔBOD vps37Δ (CTY21), bro1ΔBOD mvb12Δ (CTY22), bro1ΔBOD vps22Δ (CTY24), bro1ΔBOD snf7Δ (CTY12), bro1ΔBOD vps24Δ (CTY18), bro1ΔBOD vps2Δ (CTY26), bro1ΔBOD vta1Δ (CTY27), 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. (C) WT (SEY6210), GOY65, and GOY65 cells transformed with BRO1 and BRO1p-bro1ΔBOD were analyzed by live-cell fluorescence microscopy and quantified for the frequency of cells able to support NBD-PC trafficking to the vacuolar lumen. Error bars indicate SEM. Asterisk indicates statistically significant differences compared with WT (P = 0.0002). (D) Lysates generated from bro1Δ (GOY65) transformed with empty vector, BRO1, and BRO1p-bro1ΔBOD were analyzed by immunoblotting using antibodies against Bro1 and Pgk1. Numbers below the Bro1 blot indicate expression levels normalized to BRO1 expression. (E) WT (SEY6210), bro1ΔBOD (CTY2), and bro1V (CTY4) were analyzed by ET and quantified to assess ILV diameter. 337 ILVs from WT (SEY6210), 831 ILVs from bro1ΔBOD (CTY2), and 225 ILVs from bro1V (CTY4) were quantified. Data are represented as mean ± SEM. Data are represented as mean ± SEM. (F) WT (SEY6210), bro1Δ (GOY65), or bro1V (bro1Δ::TEF1p-bro1V; CTY4) cells were transformed with the indicated GFP-tagged cargo plasmid to assess MVB sorting using live-cell fluorescence microscopy. White dashed lines indicate cell boundaries. Scale bars = 5 µm.
Figure S3.

bro1ΔBOD-supported ILV formation requires the Vps4–ESCRT machinery. This figure complements Fig. 3. (A) Lysates generated from WT (SEY6210), vps4Δ (MBY3), bro1Δ (GOY65), TEF1p-BRO1 (CTY1), bro1ΔBOD (370–844; bro1Δ::TEF1p-bro1ΔBOD; CTY2), bro1ΔBODΔUBD (388–844; bro1Δ::TEF1p-bro1ΔBODΔUBD; CTY3), TEF1p-bro1V (CTY4), and bro1ΔBOD vps4Δ (CTY5) cells were analyzed by immunoblotting using antibodies against Bro1, Vps4, and Pgk1. Numbers below the Bro1 blot indicate expression levels normalized to BRO1 expression. (B) WT (SEY6210), bro1ΔBOD (370–844; CTY2), bro1ΔBODΔUBD (388–844; CTY3), bro1ΔBOD hse1Δ (CTY30), bro1ΔBOD vps27Δ (CTY29), bro1ΔBOD vps37Δ (CTY21), bro1ΔBOD mvb12Δ (CTY22), bro1ΔBOD vps22Δ (CTY24), bro1ΔBOD snf7Δ (CTY12), bro1ΔBOD vps24Δ (CTY18), bro1ΔBOD vps2Δ (CTY26), bro1ΔBOD vta1Δ (CTY27), 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. (C) WT (SEY6210), GOY65, and GOY65 cells transformed with BRO1 and BRO1p-bro1ΔBOD were analyzed by live-cell fluorescence microscopy and quantified for the frequency of cells able to support NBD-PC trafficking to the vacuolar lumen. Error bars indicate SEM. Asterisk indicates statistically significant differences compared with WT (P = 0.0002). (D) Lysates generated from bro1Δ (GOY65) transformed with empty vector, BRO1, and BRO1p-bro1ΔBOD were analyzed by immunoblotting using antibodies against Bro1 and Pgk1. Numbers below the Bro1 blot indicate expression levels normalized to BRO1 expression. (E) WT (SEY6210), bro1ΔBOD (CTY2), and bro1V (CTY4) were analyzed by ET and quantified to assess ILV diameter. 337 ILVs from WT (SEY6210), 831 ILVs from bro1ΔBOD (CTY2), and 225 ILVs from bro1V (CTY4) were quantified. Data are represented as mean ± SEM. Data are represented as mean ± SEM. (F) WT (SEY6210), bro1Δ (GOY65), or bro1V (bro1Δ::TEF1p-bro1V; CTY4) cells were transformed with the indicated GFP-tagged cargo plasmid to assess MVB sorting using live-cell fluorescence microscopy. White dashed lines indicate cell boundaries. Scale bars = 5 µm.

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