ATPase activities of various streptavidin-purified samples.

<p><b>(A)</b> Coomassie Blue staining after SDS-PAGE of purified wild-type complex (D^WT-C), D560N variant (D^D560N-C), E342Q variant (D^E342Q-C), and wild-type Drs2p expressed alone. <b>(B–C and E–F</b>) The ATPase activity of the same samples (after 5-fold dilution resulting in about 60 µg/mL Drs2p in the case of the WT enzyme) was measured at 30°C in a KNG medium supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 1 mM Mg-ATP, in the absence (circles and dashed lines) or presence (triangles and continuous lines) of 0.025 mg/mL PI4P. The dotted line in panels C-F is given for easier comparison with results for WT. <b>(D)</b> Functional complementation of the temperature-sensitive phenotype of <i>Δdrs2</i> yeast cells. Yeast cells, either wild-type or <i>Δdrs2</i>, were transformed with plasmids bearing <i>DRS2</i> tagged at its 5′ end, either WT or E342Q. Cells transformed with an empty vector (EV) were used as negative control. Serial dilutions of yeast cells were spotted on plates and incubated at the restrictive temperature of 20°C.