Figure 6.
Bro1 V domain mutations disrupting Vps4 stimulation in vitro disrupt MVB sorting in vivo. (A)bro1Δ (GOY65) cells were transformed with empty plasmid (pRS414) or plasmids with the BRO1 promoter and BRO1, bro1M4, bro1M8, bro1M9, or bro1M10. Localizations of model MVB cargo GFP-Cps1, Ub-GFP-Cps1, or Sna3-GFP were determined using live-cell fluorescence microscopy to assess MVB sorting in these mutant contexts. White dashed lines indicate cell boundaries. Scale bars = 5 µm. (B) The percentage of cells with WT sorting signal was quantified and calculated from at least 100 cells from three independent experiments performed on three different days from three different transformations. (C) Representative immunoblots showing mutant protein expression levels, probing against Bro1 and Pgk1 as a loading control, using lysates of GOY65 transformed with empty plasmid (pRS414) or plasmids with the BRO1 promoter and BRO1, bro1M4, bro1M8, bro1M9, or bro1M10.

Bro1 V domain mutations disrupting Vps4 stimulation in vitro disrupt MVB sorting in vivo. (A) bro1Δ (GOY65) cells were transformed with empty plasmid (pRS414) or plasmids with the BRO1 promoter and BRO1, bro1M4, bro1M8, bro1M9, or bro1M10. Localizations of model MVB cargo GFP-Cps1, Ub-GFP-Cps1, or Sna3-GFP were determined using live-cell fluorescence microscopy to assess MVB sorting in these mutant contexts. White dashed lines indicate cell boundaries. Scale bars = 5 µm. (B) The percentage of cells with WT sorting signal was quantified and calculated from at least 100 cells from three independent experiments performed on three different days from three different transformations. (C) Representative immunoblots showing mutant protein expression levels, probing against Bro1 and Pgk1 as a loading control, using lysates of GOY65 transformed with empty plasmid (pRS414) or plasmids with the BRO1 promoter and BRO1, bro1M4, bro1M8, bro1M9, or bro1M10.

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