Phosphorylation and dephosphorylation properties of the purified Drs2p-Cdc50p complex.

<p><b>(A)</b> Time course of ATP-dependent phosphorylation of the purified Drs2p-Cdc50p complex at various concentrations of [γ-³²P]ATP. The streptavidin-purified and Ni²⁺-TED-treated sample, diluted to about 50 µg/mL in SSR on ice in the presence of 1 mg/mL DDM and 0.05 mg/mL POPS, was supplemented with [γ-³²P]ATP (typically 4 µL of concentrated ATP was added to 40 µL enzyme) and brought to 30°C for various periods before acid quenching and filtration. <b>(B)</b> ATP-dependence of the maximal phosphorylation level, in the absence (open circles) or presence (open triangles) of PI4P at 0.025 mg/mL. In some cases, the purified sample had been first pre-incubated with 1 mM vanadate (grey symbols). <b>(C and D)</b> The streptavidin-purified sample (here before Ni²⁺-TED treatment, and diluted to about 60 µg/mL) was supplemented with 0.5 µM [γ-³²P]ATP in the absence <b>(C)</b> or presence <b>(D)</b> of PI4P, and phosphorylation at 30°C was measured (white symbols). Grey symbols refer to experiments performed in the presence of vanadate. To trigger ATP-induced chase of the radioactively-labelled phosphoenzyme, 1 mM non-radioactive Mg-ATP was added after 1.5 minute (black symbols, in the absence of vanadate).