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. 2021 Jun 23;220(8):e202102070. doi: 10.1083/jcb.202102070

Figure 2.

Figure 2.

Bro1ΔBOD does not support efficient MVB cargo sorting. (A) WT (SEY6210), bro1Δ (GOY65), or bro1ΔBOD (bro1Δ::TEF1p-bro1ΔBOD; CTY2) cells were transformed with the indicated GFP-tagged cargo plasmid to assess MVB sorting using live-cell fluorescence microscopy. Percentage of cells with WT sorting signal was quantified and calculated from at least 183 cells from four independent experiments performed on four different days from three different transformations. White dashed lines indicate cell boundaries. Scale bars = 5 µm. (B and C) Representative immunoblots showing processing of Sna3-GFP (B) or Mup1-GFP (C), including liberated GFP and ubiquitylated species in WT (SEY6210), vps4Δ (MBY3), bro1Δ (GOY65), bro1ΔBOD (bro1Δ::TEF1p- bro1ΔBOD ; CTY2), bro1ΔBOD snf7Δ (CTY12), or bro1ΔBOD vps4Δ (CTY5) cells. Pgk1 served as loading control. Data were quantified from four independent experiments performed on four separate days from two transformations. Data are represented as mean ± SD. Asterisks indicate a statistically significant difference (P < 0.05; B: BRO1∆ vs. VPS4∆ P = 0.04, BRO1∆ vs. bro1∆BOD P = 0.008; C: BRO1∆ vs. VPS4∆ P = 0.0006, BRO1∆ vs. bro1∆BOD P = 0.0255), and number signs indicate a statistically significant difference compared with WT (P < 0.0001).