Fig. 6.

Inositol polyphosphates interact with the VTC domain of Vtc4. (A) The interaction between a truncated version of Vtc4, His-Vtc4*, and reagents 1 and 2 was confirmed. Reagents 1, 2, and C were exposed to 20 μg of His-Vtc4*, incubated for 2 h at 4 °C, and eluted with either 10 mM InsP₆ or 10 mM 5PCP-InsP₅ solution (PCP). His-Vtc4* input (lane 1), supernatants (lanes 2–4), and eluents (elution with InsP₆: lanes 5–7; elution with 5PCP-InsP₅, lanes 8–10) were separated by SDS/PAGE and visualized by using silver staining. (B) Vtc4 is not pyrophosphorylated. Recombinant GST-Vtc4 was expressed and purified from S. cerevisiae and treated with 1 μM β[³²P]5PP-InsP₅ (15 μCi) in 25 mM Tris (pH 7.4, 50 mM NaCl, 6 mM MgCl₂, and 1 mM DTT) at 37 °C for 40 min, while still on beads. GST-Nsr1 and GST-Puf6 were included as positive control experiments.