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.</p

Size-exclusion chromatography and mass spectrometry analysis of the purified Drs2p-Cdc50p complex.

<p><b>(A)</b> Top: calibration of the TSK-3000SW silica gel column with gel filtration standards. The elution volume of Thyroglobulin corresponds to the dead volume (V₀), while Vitamin B12 is eluted at a volume close to the total volume (V_t). Bottom: size-exclusion chromatography profile of the streptavidin-purified Drs2p-Cdc50p complex (continuous line). The streptavidin-purified and Ni²⁺-TED-treated fraction was first concentrated on YM100 filters and 300 µL was then injected on the column. Fractions 1–4 were collected. The dotted line shows the behavior of a control DDM-solubilized SR SERCA1a sample (500 µL at 4 mg/mL was injected). <b>(B)</b> Analysis of the collected fractions on an 8% Coomassie Blue stained SDS-PAGE. <b>(C)</b> An aliquot of the SEC-eluted sample was submitted to mass spectrometry analysis. Spectra were acquired on a range of m/z values from 30,000 to 200,000.</p

Streptavidin-based purification of Drs2p and Cdc50p.

<p>N-terminally Bad-tagged Drs2p and His₁₀-tagged Cdc50p were purified onto a streptavidin column, and eluted by TEV cleavage. <b>(A)</b> Coomassie Blue stained 10% SDS-PAGE. <b>(B, C, D)</b> Immunodetection using a Biotin probe, a α-Drs2p antibody and a Histidine probe, as indicated. T, total membranes before solubilization; S, DDM-solubilized fraction; FT, flow-through; R, Streptavidin Sepharose resin before addition of TEV; R_TEV, Streptavidin Sepharose resin after incubation with TEV for 16 h at 6°C; E, fraction eluted from the resin after incubation with TEV. Drs2p_(t), truncated form of Drs2p; Cdc50p_(g), glycosylated form of Cdc50p; His₆-TEV, N-terminally hexahistidine-tagged TEV.</p