Figure 1.
MYM-type zinc finger containing proteins ZNF261 and ZNF198 have SUMO-binding activity.
A: Schematic diagrams of MYM-type zinc finger containing proteins. Amino acid number is shown next to each schematic diagram. Locations of the myeloproliferative and mental retardation-type zinc fingers (MYM-type zinc fingers, gold), proline/valine-rich domains (P/V-rich, green), and Cre-like domains (CL domain, blue) are shown. The MYM-type zinc finger consensus motif where X is any amino acid is shown below. B: Full-length ZNF261 or ZNF198 proteins were in vitro transcribed and translated in the presence of [35S] methionine and bound to immobilized GST-tagged SUMO-1, SUMO-2 or SUMO-2(x3). Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. Binding to GST alone was performed as a negative control. All binding assays contained equivalent amounts of GST and GST-tagged SUMO proteins as determined by immunoblot analysis of eluted proteins with an anti-GST antibody.
Figure 2.
MYM-type zinc fingers in ZNF261 and ZNF198 are involved in SUMO-binding.
A: GST and GST-SUMO-2(x3) were immobilized on glutathione coated plates and incubated with 35S-labeled full-length or N- or C-terminus truncation fragments of ZNF261. Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. B: Pyruvate kinase or pyruvate kinase fused to the MYM-type zinc fingers from ZNF261 or ZNF198 were incubated with immobilized GST (white), GST-SUMO-2 (grey), and GST-SUMO-2(x3) (black). Unbound proteins were removed by washing and bound proteins were determined by liquid scintillation detection of eluted proteins. Plotted values represent the mean +/− S.D. from three independent experiments. C: ZNF261 was run at three concentrations in the analytical ultracentrifuge (10, 20, and 40 µM). Representative sedimentation data from the 20 µM ZNF261(1-495) data set is shown in the left panel. The data was globally fit to a single species model and the residuals between the calculated and experimental absorbance are shown below. The residuals appear randomly scattered around zero indicating that a single species model describes the data. ZNF261(1-495) (20 µM) was run with three concentrations of SUMO-2(x2) (20, 40, and 80 µM) in the analytical ultracentrifuge. Representative sedimentation data of the 20 µM ZNF261 and 80 µM SUMO-2(x2) data set is shown in the right panel. Data were globally fit to an A+B → AB model and the residuals between the calculated and experimental absorbance are shown below. The global reduced chi-squared value was 3.13.
Figure 3.
MYM-type zinc fingers interact with the α-helix and second β-strand in SUMO-2.
In vitro expressed full-length ZNF261 and ZNF198 were incubated with GST or GST-tagged SUMO-2, SUMO-2(QFI), SUMO-2(x3), and SUMO-2(x3)(QFI). Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. All binding assays contained equivalent amounts of GST and GST-tagged SUMO proteins as determined by immunoblot analysis of eluted proteins with an anti-GST antibody.
Table 1.
ICP-MS analysis of zinc content in ZNF261(1-495).
Figure 4.
Zinc chelation does not significantly perturb SUMO-binding activity of ZNF261.
A: In vitro expressed full-length ZNF261 proteins were incubated overnight with immobilized GST or GST-tagged SUMO-2(x3) in assay buffer (untreated) or assay buffer containing 50 mM EDTA/100 mM sodium acetate, pH 5.5 (EDTA treated), or 1,10-phenanthroline (1,10-phen), or 1,7-phenanthroline (1,7-phen). Unbound proteins were washed away and bound proteins were visualized by SDS-PAGE followed by autoradiography. B: Immobilized GST or GST-tagged SUMO-2(x3) was incubated with 35S-labeled ZNF261(1-495) wild-type or ZNF261(1-495) containing cysteine to alanine substitutions. Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography.
Figure 5.
The N-terminus and MYM-type zinc fingers in ZNF198 are required for localization to PML-NBs.
A: U2OS cells were transfected with constructs encoding myc-tagged ZNF198 full-length or truncation fragments. Localization of ZNF198 with PML-NBs was analyzed by fluorescence microscopy. B: Full-length and truncation fragments of myc-tagged ZNF198 were expressed in U2OS cells, immunopurified with anti-myc agarose beads, and detected with anti-myc and anti-SUMO-2 antibodies. Full-length protein bands are marked with an asterisk in each input lane. C: Myc-tagged ZNF198 and FLAG-tagged PML were co-expressed in U2OS cells, immunopurified with anti-myc agarose beads or anti-FLAG agarose beads, and detected with anti-myc and anti-PML antibodies.