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. 2006;34(18):5007-20.
doi: 10.1093/nar/gkl575. Epub 2006 Sep 20.

ZBP1 Subcellular Localization and Association With Stress Granules Is Controlled by Its Z-DNA Binding Domains

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Free PMC article

ZBP1 Subcellular Localization and Association With Stress Granules Is Controlled by Its Z-DNA Binding Domains

Nikolaus Deigendesch et al. Nucleic Acids Res. .
Free PMC article

Abstract

Z-DNA binding protein 1 (ZBP1) belongs to a family of proteins that contain the Zalpha domain, which binds specifically to left-handed Z-DNA and Z-RNA. Like all vertebrate proteins in the Zalpha family, it contains two Zalpha-like domains and is highly inducible by immunostimulation. Using circular dichroism spectroscopy and electrophoretic mobility shift assays we show that both Zalpha domains can bind Z-DNA independently and that substrate binding is greatly enhanced when both domains are linked. Full length ZBP1 and a prominent splice variant lacking the first Zalpha domain (DeltaZalpha) showed strikingly different subcellular localizations. While the full length protein showed a finely punctate cytoplasmatic distribution, ZBP1DeltaZalpha accumulated in large cytoplasmic granules. Mutation of residues important for Z-DNA binding in the first Zalpha domain resulted in a distribution comparable to that of ZBP1DeltaZalpha. The ZBP1DeltaZalpha granules are distinct from stress granules (SGs) and processing bodies but dynamically interacted with these. Polysome stabilization led to the disassembly of ZBP1DeltaZalpha granules, indicating that mRNA are integral components. Heat shock and arsenite exposure had opposing effects on ZBP1 isoforms: while ZBP1DeltaZalpha granules disassembled, full length ZBP1 accumulated in SGs. Our data link ZBP1 to mRNA sorting and metabolism and indicate distinct roles for ZBP1 isoforms.

Figures

Figure 1
Figure 1
Comparison of Zα domains (A) and schematic representation of constructs used for transfections (B) and protein expression (C). Comparison of Zα domains of human (hs) and mouse (mm) ZBP1, human ADAR1, zebrafish (dr) PKZ, vaccinia virus (vv) E3L and yaba-like disease virus (yldv) E3L is shown (A). The structures of the mouse (mm) ZαZBP1, human (hs) ZαADAR1, yaba-like disease virus (yldv) ZαE3L domains have been determined in complex with Z-DNA. Residues that make contact with Z-DNA, or the analogous residues in other Zα domains, are boxed in light blue. Asterisks mark the conserved asparagine and tyrosine residues that have been mutated in this study in hsZBP1, as well as a conserved tryptophan. Residues that form the hydrophobic core are boxed in green. Residues that are neither DNA contacting nor structural but match the consensus sequence are highlighted in yellow. Isoleucine 335 in ZβADAR1 is highlighted in red. (B) The exon composition of the most prominent ZBP1 splice variants ZBP1full and ZBP1ΔZα as well as that of artificial constructs are shown. Exon 7 is rarely found in mRNA. Exon 9 contains an alternative termination site (22). ZBP1full and ZBP1ΔZα have been expressed as un-tagged or GFP tagged proteins in HeLa cells. ZBP1ΔZβ, ZBP1ΔZαΔZβ, ZBP1E1-5 and ZBP1E1-5ΔZα were expressed as GFP-tagged proteins. Schematic representation of the exon composition of constructs expressed from pET28a (p28) vectors in E.coli are shown in (C).
Figure 2
Figure 2
SDS–PAGE analysis of recombinant proteins used for CD-spectrometry and EMSA. 500 nmol of each purified protein was subjected to SDS–PAGE and visualized by Coomassie Brilliant blue staining: Lane M, molecular mass marker; lane 1: ZαβADAR1; lane 2: ZαZBP1; lane 3: ZβZBP1; lane 4: ZαβZBP1; lane 5: ZαβZBP1N46D; lane 6: ZαβZBP1Y50A; lane 7: ZαβZBP1N46D/Y50A; lane 8: ZαβZBP1N141D/Y145A; lane 9: ZαβZBP1 N46D/Y50A/N141D/Y145A.
Figure 3
Figure 3
Conformational change of poly(dC–dG) in the presence of Zα proteins in CD-spectrometry. The CD spectra show the titrations of ZαZBP1 (A), ZβZBP1 (B), ZαβZBP1 (C) and ZαβADAR1 (D). The curves show DNA spectra in the absence (control, B-DNA) and in the presence of protein at protein/basepair molar ratio as labeled. Spectra were measured after 30 min of incubation at 24°C. (E) The CD spectra of wildtype (wt) ZαβZBP1 and quadruple mutant (ZαβZBP1mut) carrying the four amino acid substitutions N46D, Y50A, N141D and Y145A are shown in relation to the control, where no protein was added. (F) Time dependent change of ellipticity at 255 nm wavelength over 30 min ZαβZBP1 and ZαβZBP1mut are shown. Spectra are expressed in absolute values of ellipticity in millidegrees (mdeg).
Figure 4
Figure 4
Binding of Z-DNA by ZBP1 Zα proteins in electrophoretic mobility shift assays. EMSA were performed using 32P-labeled double-stranded (dC–dG)20 as probe in the presence of 20 000-fold access of sheared salmon sperm DNA as unspecific competitor. (A) Molar concentration was adjusted for the number of Zα domains the protein comprises. Zα and Zβ was assayed in twice as high molar concentration as Zαβ. Migration of the free probe is shown in lane 1 and 13. Zαβ (lane 2: 12.5 nM; lane 3: 25 nM; lane 4: 50 nM), Zα (lane 5: 25 nM; lane 6: 50 nM; lane 7: 100 nM), and Zβ (lane 5: 25 nM; lane 6: 50 nM; lane 7: 100 nM) were assayed at indicated concentrations. Reaction mixes shown in lanes 11–13 were prepared at the same time and were run on a separate TBE Gel. Zα and Zβ were mixed together at two concentrations (lane 11: 25 nM of each protein and lane 12: 50 nM of each protein) to see if there is a cooperative effect if both domains are present in different molecules. Sometimes a minor, slower migrating band was observed (asterisk) for the probe, probably due to a different secondary structure of the probe. (B) ZαβADAR1 (lane 2) and ZαβZBP1 (lane 3) form complexes with the radiolabeled probe whereas the indicated ZαβZBP1 mutants at equimolar concentration do not (lanes 4–8). Migration of the free probe is shown in lane 1.
Figure 5
Figure 5
Different subcellular localization of two major ZBP1 splice variants. HeLa cells transiently transfected with ZBP1 were fixed and stained with polyclonal anti-ZBP1 antibodies and with secondary RPE-conjugated anti-rabbit-Ig antibodies. The left pictures show red fluorescence (RPE) after identical exposure time. The middle panels show nuclear stain using Hoechst33342. Right pictures show merges. In (A) no competitor was present, while in (B) 10 μg/ml of recombinant ZαβZBP1 was added. Anti-ZBP1 antibodies were used to stain for ZBP1 (left panels) in HeLa cells transfected with either full length ZBP1 (C–E) or the splice variant ZBP1ΔZα (F–H). Cells representative of three independent experiments are shown.
Figure 6
Figure 6
Correlation of subcellular localization of ZBP1 with the capability of the first Zα domain to bind the Z-conformation. HeLa cells were transiently transfected with the indicated ZBP1-EGFP expression plasmids for 8–12 h. Subcellular localization was analyzed by fluorescence microscopy (left panels). Nuclei were stained with Hoechst33342 (middle panel). In (A) and (B) localization of the naturally occurring full length ZBP1 and ZBP1ΔZα, respectively, tagged with GFP is shown. Localization is shown for GFP-tagged full length ZBP1 with the indicated single (C and D), double (E and F) or quadruple (G) amino acid substitutions at residues important for Z-DNA binding. (H and I) show the localization of full length ZBP1 and ZBP1ΔZα proteins, respectively, after the precise deletion of exon 4, which encodes the entire second Zα domain. The localization of fusion proteins after deletion of the complete C-terminus (exons 6–10) is shown for ZBP1 (J) and ZBP1ΔZα (K). (L) shows the distribution of GFP alone within the cell. Two hours after transfection with full length ZBP1-GFP (M) and ZBP1ΔZα-GFP (N) constructs, cells were treated with Leptomycin B, an inhibitor of nuclear export, for 8 h prior to microscopy. Cells representative of at least three independent experiments are shown. (O) Western blot analysis shows that ZBP1-GFP fusion proteins were intact and not significantly degraded 10 h after transfection. Total cell lysates of transfected cells were subjected to SDS–PAGE and recombinant proteins were detected with an antibody directed against GFP. In lane 1 lysate of mock-transfected cells is shown as control. Lane 2 shows lysate of GFP transfected cells. The letters written in parenthesis for lanes 3–9 refer to the different fusion proteins that are shown in this figure as fluorescence pictures (A–K).
Figure 7
Figure 7
Association and dynamics of interaction of ZBP1 granules with SGs and PBs. HeLa cells were co-transfected with full length ZBP1-RFP (A), ZBP1ΔZα-RFP (B and C), full length ZBP1-GFP (D) and ZBP1ΔZα-GFP (E) plus SG markers G3BP-GFP (A and B), TIA1-GFP (C) or PB marker DCP1a-RFP (D and E). For each photo the indicated sections are shown in better resolution below for green, red and merged fluorescence. The dynamics of interaction between ZBP1 granules with SGs and PBs are shown for co-transfections with ZBP1ΔZα-RFP and TIA1-GFP (F and Video 1), ZBP1ΔZα-RFP and G3BP-GFP (Video 2) or ZBP1ΔZα-GFP and DCP1a-RFP (G and Videos 3 and 4). Cells were observed over a period of 15 min with pictures taken every 30 s. Pictures taken at 3 min intervals are shown in F and G. White arrows indicate ZBP1 granules that were unbound at the beginning of the observation period and later attached to SGs (F) or PBs (G). Yellow arrows indicate ZBP1 granules that stayed attached to SGs (F) or PBs (G) over the complete observation period. Cells representative of at least three independent experiments are shown.
Figure 8
Figure 8
Relocation of full length ZBP1 and ZBP1ΔZα upon arsenite treatment and heat shock. HeLa cells were co-transfected with G3BP-GFP and full ZBP1-RFP (AD) or ZBP1ΔZα (EH). After 7 h cells were incubated with 100 μg/ml emetine (B and F) or 500 μM arsenite for 1 h or exposed to heat shock at 44°C for 30 min (D and H). Upper panels show RFP fluorescence, middle panels GFP fluorescence and lower panels the merged views. Cells representative of at least three independent experiments are shown. Supplementary Data.

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