FIGURE 4.

Formation of the Drs2p phosphoenzyme intermediate is dependent on Cdc50p. A, to purify 100% Cdc50p-bound and monomeric Drs2p, the Ni²⁺-NTA eluate (E_NTA) fraction obtained in Fig. 2A was subjected to a second affinity purification step in which α-Myc antibody-coupled beads were used to capture all Drs2p molecules associated with Cdc50p-Myc. The flow-through (FT_MYC) contained primarily monomeric Drs2p (less than 1% Cdc50p-bound). The molecules bound to the α-Myc-coupled beads were eluted using a triple Myc peptide, yielding 100% Cdc50p-bound Drs2p (E_MYC). That this elution step did not dissociate the complex was verified by a subsequent binding step to Ni²⁺-NTA beads. All fractions were analyzed by immunoblotting, using α-HA and α-Myc antibodies. FT_HA, flow-through α-HA beads; E_HA, α-HA bead eluate; FT_NTA, flow-through Ni²⁺-NTA beads. B, equal amounts of 100% Cdc50p-bound and monomeric H₂-Drs2p were labeled with 2 μm [γ-³²P]ATP at 4 °C for 50 s in the absence or presence of 1 mm VO₄. The VO₄-sensitive phosphoenzyme formation is reported on the y axis. Data presented are means ± S.D. (error bars) of three independent experiments. C, dephosphorylation of 100% Cdc50p-bound Drs2p, triggered by the addition of either 5 mm ATP or 5 mm ADP as described in the legend for Fig. 3. Error bars) indicate S.D.