Figure S4.

Bro1V mutants bind Vps4. This figure complements Figs. 5 and 6. (A) Immobilized His₆-Vps4 or Ni-NTA beads alone were incubated with Bro1V WT or Bro1V mutants, and bound material was analyzed by immunoblotting with anti-Bro1 antiserum. M, mutant. (B) Mutant protein expression levels normalized to WT. Bro1 expression levels were normalized to Pgk1 and subsequently normalized to WT/Pgk1 ratios. Quantified from three independent experiments performed on three different days from two sets of transformations. Data are represented as mean ± SEM. Immunoblots were probed against Bro1 and PGK1 using lysates of GOY65 transformed with empty plasmid (pRS414) or BRO1, bro1^M4, bro1^M8, bro1^M9, or bro1^M10 plasmids. Statistical analyses did not reveal differences between WT and mutant forms of Bro1. (C) bro1Δ (GOY65) cells were transformed with GFP-Cps1 and vector, BRO1, BRO1^M1, or BRO1^M7 plasmids to assess the impact of these mutations on GFP-Cps1 sorting. White dashed lines indicate cell boundaries. Scale bar = 5 µm.