Characterization of the SUMO-Binding Activity of the Myeloproliferative and Mental Retardation (MYM)-Type Zinc Fingers in ZNF261 and ZNF198

<div><p>SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM)-type zinc fingers. We demonstrated that MYM-type zinc fingers from ZNF261 and ZNF198 are necessary and sufficient for SUMO-binding and that individual MYM-type zinc fingers function as SUMO-interacting motifs (SIMs). Our binding studies revealed that the MYM-type zinc fingers from ZNF261 and ZNF198 interact with the same surface on SUMO-2 recognized by the archetypal consensus SIM. We also present evidence that MYM-type zinc fingers in ZNF261 contain zinc, but that zinc is not required for SUMO-binding. Immunofluorescence microscopy studies using truncated fragments of ZNF198 revealed that MYM-type zinc fingers of ZNF198 are necessary for localization to PML-nuclear bodies (PML-NBs). In summary, our studies have identified and characterized the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198.</p></div

Zinc chelation does not significantly perturb SUMO-binding activity of ZNF261.

<p>A: <i>In vitro</i> 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>B</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.</p

The N-terminus and MYM-type zinc fingers in ZNF198 are required for localization to PML-NBs.

<p>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>B</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. <b>C</b>: 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.</p

MYM-type zinc finger containing proteins ZNF261 and ZNF198 have SUMO-binding activity.

<p>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>B</b>: Full-length ZNF261 or ZNF198 proteins were <i>in vitro</i> 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.</p

MYM-type zinc fingers interact with the α-helix and second β-strand in SUMO-2.

<p><i>In vitro</i> 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.</p

MYM-type zinc fingers in ZNF261 and ZNF198 are involved in SUMO-binding.

<p>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>B</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. <b>C</b>: 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.</p

A high throughput mutagenic analysis of yeast sumo structure and function

<div><p>Sumoylation regulates a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using <i>S</i>. <i>cerevisiae</i>, a model organism with a single essential SUMO gene (<i>SMT3</i>), we developed a library of >250 mutant strains with single or multiple amino acid substitutions of surface or core residues in the Smt3 protein. By screening this library using plate-based assays, we have generated a comprehensive structure-function based map of Smt3, revealing essential amino acid residues and residues critical for function under a variety of genotoxic and proteotoxic stress conditions. Functionally important residues mapped to surfaces affecting Smt3 precursor processing and deconjugation from protein substrates, covalent conjugation to protein substrates, and non-covalent interactions with E3 ligases and downstream effector proteins containing SUMO-interacting motifs. Lysine residues potentially involved in formation of polymeric chains were also investigated, revealing critical roles for polymeric chains, but redundancy in specific chain linkages. Collectively, our findings provide important insights into the molecular basis of signaling through sumoylation. Moreover, the library of Smt3 mutants represents a valuable resource for further exploring the functions of sumoylation in cellular stress response and other SUMO-dependent pathways.</p></div

Analysis of <i>smt3</i> SIM binding mutant alleles.

<p>(A) The identified lethal and conditional <i>smt3</i> mutants found within the SIM binding surface mapped onto the β2-α1 region of Smt3 (PDB: 1EUV). Residues that gave rise to lethal and conditional phenotypes are boxed in red and magenta, respectively. (B) SIM binding mutants are stress sensitive. The indicated strains were cultured overnight and serially diluted and spotted onto plates with or without hydroxyurea (HU). The plates were incubated at 30°C or 39°C, as indicated. (C) The <i>smt3</i> I35A mutant allele accumulates unusual conjugates. Wild type and <i>smt3</i> I35A strains were grown to mid-log phase, diluted 4 fold then shifted to 39°C for 20 hrs. Samples were collected at 0 and 20 hr time points and analyzed by immunoblot analysis. The stacking portion of the gel was left intact so that ultra-high molecular mass conjugates could be visualized. (D) The <i>smt3</i> I35A mutant allele exhibits cell cycle defects. Wild type and <i>smt3</i> I35A strains were grown to mid-log phase at 30°C and then shifted to 39°C for 20 hrs. Cells were collected at 20 hrs, permeabilized and stained with DAPI. Each bar represents the average of 3 independent experiments in which at least 150 cells were counted per experiment. Vertical bars indicate the standard error. Astericks denote a p-value < 0.05. (E) Phosphorylation of T42 and T43 is not critical for <i>smt3</i> function. The <i>smt3Δ</i> shuffle strain was transformed with plasmids coding for the indicated <i>Smt3</i> alleles. The transformants were grown overnight, serially diluted and spotted onto selective media in the presence or absence of 5-FOA at 30°C and 37°C.</p

Analysis of <i>smt3</i> K/R mutant alleles.

<p>(A) and (B) Strains expressing the indicated <i>smt3</i> alleles were grown overnight, serially diluted then spotted onto plates in the presence or absence of HU. The plates were incubated at 30°C or 39°C, as indicated.</p

Analysis of lethal <i>smt3</i> mutant alleles.

<p>(A) The identified lethal mutations mapped onto the Smt3 crystal structure (PDB: 1EUV). Mutated residues giving rise to lethal phenotypes are highlighted red. Also highlighted are residues predicted or known to be important for conjugation (yellow), deconjugation (blue), conjugation and deconjugation (green) or SIM binding (orange). Lethal mutations in residues important for conjugation, deconjugation or SIM binding are boxed with appropriate corresponding colors. *R71E and G98A mutations were episome remedial. (B) Analysis of the expression and conjugation profiles of lethal <i>smt3</i> mutant alleles. Mutant alleles were expressed in a SUMO1 integrated strain and then analyzed by immunoblot analysis. The asterisk and high molecular mass species seen in the vector only control (also in C and D) represent non-specific, cross-reacting proteins. (C) Analysis of deconjugation in response to ATP depletion. Lethal mutant alleles that form ultra-high molecular mass conjugates were expressed in a SUMO1 integrated strain. Cultures at mid-log phase were grown in normal medium (-) or ATP depletion medium (+) containing sodium azide and 2-deoxyglucose for 10 minutes. Cell lysates were analyzed by immunoblot analysis. (D) Analysis of deconjugation and conjugation following ATP depletion and restoration. The lethal mutants not forming ultra-high molecular mass conjugates were expressed in a SUMO1 integrated strain. Cultures at mid-log phase were grown in normal (-) or ATP depletion media (+) for 10 minutes. Cells were then allowed to recover for 10 minutes in normal medium (+’). Cell lysates were analyzed by immunoblot analysis. (E) Analysis of Smt3 protein localization. The indicated Smt3 proteins were expressed by transforming a SUMO1 expressing strain with the indicated constructs. Transformants were grown to mid-log phase, fixed, spheroplasted and permeabilized. Smt3 localization was determined by immunofluorescence microscopy. DNA was labeled with DAPI. (F) Co-localization of Smt3 foci and Cdc48. A GFP-Cdc48 expressing strain was transformed with empty vector or vectors coding for wild type or F65A mutant Smt3. Transformants were grown to mid-log phase and fixed, spheroplasted and permeabilized. Smt3 and GFP-Cdc48 localization were determined by immunofluorescence microscopy. DNA was labeled with DAPI.</p