Fig. 1

Figure 1. Nudt3 is a Zn²⁺-dependent endo-polyPase

(A) Heparin affinity fractionation step. After tissue homogenization, cell lysis, and centrifugation, the supernatant was loaded into a heparin-based affinity chromatography column. Proteins were eluted by applying a NaCl-based ionic strength gradient (dashed line). Endo-polyPase activity was assayed using the indicated divalent cations (see STAR Methods section).

(B) PolyP-zymogram and mass spectrometry identification. The left panel shows a proof of concept for the polyP-zymogram: recombinant yeast Ddp1 was separated in a non-denaturing acrylamide gel containing polyP. polyPase activity was permitted by incubating the gel in the appropriate conditions. The gel was stained with DAPI. In the right panel, containing activity aliquot from the size exclusion column was analyzed in the polyP-zymogram as above. The area where the polyPase activity appeared was excised, and proteins were identified by tandem mass spectrometry analysis. A list of the proteins showing the higher score is provided.

(C and D) Human Nudt3 obtained from E. coli (C) and the HEK293T cell line (D) have Zn²⁺-dependent endo-polyPase activity. Nudt3 was incubated with 5 μg of polyP₁₀₀ for the indicated times in the presence of 5 mM of the noted cations, electrophoretically separated in PAGE-urea gels, and polyP was visualized using DAPI staining.

(E) Nudt3 is active at physiological pH. Nudt3 was incubated at different pH values with 5 μg of polyP₁₀₀; polyP was separated in PAGE-urea gels and stained with DAPI.

(F) Nudt3 endo-polyPase activity is inhibited by phosphatase inhibitors. As in (C), using 5 mM Zn²⁺ as a cation and the concentration of the noted inhibitors. incubation time was 1 h.

(G) Nudt3^E70A does not present endo-polyPase activity. As in (C) using 5 mM Zn²⁺.

(H) Endo-polyPase activity quantification of the different members of the human DIPP family purified from E. coli. As in (C) using 5 mM Zn²⁺. Incubation time was 1 h. Mean ± SEM of three independent experiments. ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05.

See also Figures S1 and S2.