Protein Polyphosphorylation of Lysine Residues by Inorganic Polyphosphate  Cristina Azevedo, Thomas Livermore, Adolfo Saiardi  Molecular Cell  Volume 58, Issue 1, Pages 71-82 (April 2015) DOI: 10.1016/j.molcel.2015.02.010 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 58, 71-82DOI: (10.1016/j.molcel.2015.02.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Nsr1 Is Polyphosphorylated (A) Simplified scheme of inositol pyrophosphates and polyP metabolism. Kinases are shown in blue and phosphatases in red. (B) Nsr1 mobility shift on NuPAGE. WT, vtc4Δ, vip1Δ, kcs1Δ, and ppx1Δ protein extracts were resolved and immunoblotted. (C) Nsr1 is dephosphorylated by Ppx1. WT protein extract was incubated with or without Ppx1 prior to western blotting. (D) Proposed model for Nsr1 post-translational modification, polyphosphorylation. Stroke lines are used when the figure was assembled from the same gel/membrane, whereas solid lines represent different gel/membranes. See also Figure S1. Molecular Cell 2015 58, 71-82DOI: (10.1016/j.molcel.2015.02.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Polyphosphate Is the Polyphosphorylation Effector Molecule (A) WT polyP extract is able to shift Nsr1. Purified gNsr1-13Myc from kcs1Δ (unmodified) was incubated, with polyP extract (corresponding to 15 μg of RNA) resolved and blotted. (B) PolyP is the polyphosphorylation effector. WT polyP extract was treated with recombinant Ppx1, Ddp1, DNase, or RNase. Enzymes were subsequently phenol precipitated, and the polyP extract was incubated with purified, unmodified gNsr1-13Myc (kcs1Δ). Beads were washed, resolved, and immunoblotted. (C) Chemical synthesis of polyP. Schematic representation of polyP synthesis (left). ATP was incubated in presence of recombinant EcPPK and the ATP recycling system, phosphocreatine (PC), and creatine phospho-kinase (CPK). Lane 1, overnight enzymatic reaction; lane 2, polyP > 100 kDa after removing nucleotides and enzymes with a 100-kDa filter. (D) Chemically synthesized polyP polyphosphorylates Nsr1. Purified, unmodified gNsr1-13Myc (kcs1Δ) was incubated with WT polyP; chemically synthesized polyP or chemically synthesized polyP higher than 100 kDa. Beads were washed, resolved, and immunoblotted. (E) PolyP is released from purified Nsr1 after mild acid hydrolysis. Experimental flow chart (left); purified unmodified gNsr1-13Myc (vtc4Δ) beads were incubated with chemically synthesized 32P-polyP, extensively washed, and treated with perchloric acid (PA); beads were washed, resolved, autoradiographed (middle-top), transferred, and immunoblotted (middle-bottom); supernatant was neutralized, run on a 30% PAGE, and autoradiographed (right). Polyphosphate standard of 25 phosphate length stained in toluidine was used as a marker. Stroke lines are used when the figure was assembled from the same gel/membrane, whereas solid lines represent different gel/membranes. See also Figure S2. Molecular Cell 2015 58, 71-82DOI: (10.1016/j.molcel.2015.02.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Top1 Is Polyphosphorylated (A) Top1 shows mobility shift on NuPAGE. Protein extracts from gTop1-13Myc in WT, vtc4Δ, and vip1Δ yeast were resolved and immunoblotted. (B) Top1 is polyphosphorylated by WT polyP. Purified, unmodified gNsr1-13Myc (vtc4Δ) was incubated with total WT polyP (corresponding to 15 μg of RNA). Beads were washed, resolved, and immunoblotted. (C) PolyP is released from purified Top1 after mild acid hydrolysis. Unmodified gTop1-13Myc (vtc4Δ) was incubated with 32P-polyP as above; gTop1-13Myc beads were washed. Three-eighths of the sample was resolved by PAGE, stained with stain-all and silver staining (left, middle panel). The same PAGE was subsequently dried and autoradiographed (left, left panel). One-eighth of the sample was resolved, transferred, and immunoblotted (left, right panel), and the remaining one-half of the gTop1-13Myc beads were treated under mild acidic conditions run on a 30% PAGE and autoradiographed (right). Standard of P45 phosphate length stained in toluidine was used as a marker. (D) PolyP is released, after mild acid hydrolysis, from Top1 purified from orthophosphate-labeled yeast cells. gTop1-13Myc purified from 32P-orthophosphate-labeled vip1Δ was treated with PA for 10 min at 37°C and 55°C, and supernatant was neutralized, run on a 30% PAGE, and autoradiographed (right). (E) Polyphosphate transfer involves attack in the middle of a polyP chain. See Experimental Procedures for synthesis of double-ended biotinylated P45 polyP (schematic representation on the top panel). Bottom panel: lane 1, purified, unmodified gTop1-13Myc (vtc4Δ) treated with P45 polyP that had previously been hydrolyzed with His-Ppx1; lane 2, purified, unmodified gTop1-13Myc (vtc4Δ) treated with P45 polyP, washed, and subsequently treated with His-Ppx1 to remove the shift; lane 3, purified, unmodified gTop1-13Myc (vtc4Δ) treated with P45; lane 4, purified, unmodified gTop1-13Myc (vtc4Δ); lane 5, purified, unmodified gTop1-13Myc (vtc4Δ) treated with double-ended biotinylated P45 polyP that had previously been hydrolyzed with His-Ppx1 to remove any single-ended or non-labeled polyP. Figures assembled from the same gel/membrane have stroked lines, and from different gel/membranes have solid lines. See also Figure S3. Molecular Cell 2015 58, 71-82DOI: (10.1016/j.molcel.2015.02.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Polyphosphorylation Extent Reflects PolyP Size, and Top1 Is Polyphosphorylated by Nuclear PolyP (A) PAGE and gel-shift assay of total WT polyP fractionated by gel filtration. WT polyP (230 μg of RNA) was treated with RNase and filtered through a 100-kDa membrane. The retained, molecules larger than 100 kDa, were separated by gel filtration (Superdex). A total of 10% of each fraction, pooled in pairs, was resolved by 20% PAGE and stained with DAPI (top). Purified, unmodified gNsr1-13Myc (vtc4Δ) was incubated with 10% of pooled gel filtration fractions. Beads were washed, resolved, and immunoblotted (bottom). (B) WT nuclear polyP is homogenous in size. Total WT polyP (corresponding to 4 μg of RNA) and nuclear polyP (corresponding to 30 μg of RNA) were treated with recombinant His-ScPpx1, resolved by 30% PAGE, and stained with DAPI. PolyP, 25 phosphate length, is used as a marker. (C) Top1 is polyphosphorylated by nuclear polyP. Experiment performed as in Figure 3B with nuclear polyP (corresponding to 80 μg of RNA). Molecular Cell 2015 58, 71-82DOI: (10.1016/j.molcel.2015.02.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Top1 and Nsr1 Are Polyphosphorylated on Lysine Residues (A) Top1 and Nsr1 PASK domain sequences with respective color-coded mutations. Top1 mutations were introduced between aa1 and aa149 with the remaining C-terminal protein as WT. Nsr1 mutations were introduced between aa1 and aa188 of the PASK domain. (B) Top1 K mutants show loss of mobility shift in vivo. Protein extracts from WT, vtc4Δ, and vip1Δ yeast transformed with different mutant constructs of GST-Top1 were resolved and immunoblotted. (C) Top1 K mutants are not polyphosphorylated in vitro by WT polyP. Purified, unmodified GST-Top1 (vtc4Δ) mutants were incubated with total WT polyP (corresponding to 15 μg of RNA). Beads were washed, resolved, and immunoblotted. (D) Nsr1 PASK domain K mutant is not polyphosphorylated. Purified GST-Nsr1 PASK (K-R) (vtc4Δ) beads (left) or protein extract (right) were incubated with WT polyP. Washed beads and extracts were resolved and immunoblotted. (E) Polyphosphorylation is stable under extreme alkali conditions. WT gTop1-13Myc extracts were treated in alkali conditions with increasing sodium hydroxide molarity, resolved, and immunoblotted. (F) Polyphosphorylation is very sensitive to hydroxylamine treatment. WT gTop1-13Myc extracts were treated in increasing concentrations of NH2OH as indicated, resolved, and immunoblotted. Figures assembled from the same gel/membrane have stroked lines, and from different gel/membranes have solid lines. See also Figure S4. Molecular Cell 2015 58, 71-82DOI: (10.1016/j.molcel.2015.02.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Polyphosphorylation of Nsr1 and Top1 Affects Their Interaction and Cellular Localization (A) Polyphosphorylation negatively regulates Nsr1 and Top1 interaction. Purified, unmodified Top1-13Myc (vtc4Δ) was incubated with WT polyP to immunoprecipitate Nsr1; washed beads were incubated with vtc4Δ extract in absence or presence of WT polyP; washed beads were resolved and immunoblotted. (B) Polyphosphorylation of Nsr1 affects its cellular localization. Immunofluorescence of WT, vip1Δ, kcs1Δ, and vtc4Δ with anti-Nsr1 antibody in green (anti-mouse Alexa Fluor 488) and the nucleus in blue (DAPI). (C) Polyphosphorylation of Top1 affects its cellular localization. Immunofluorescence of WT, vip1Δ, kcs1Δ, vtc4Δ, and nsr1Δ transformed with GST-Top1, stained with anti-GST antibody in red (anti-rabbit Alexa Fluor 568) and the nucleus in blue (DAPI). Scale bar, 2 μM. Molecular Cell 2015 58, 71-82DOI: (10.1016/j.molcel.2015.02.010) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 Polyphosphorylation Negatively Regulates Top1 Function (A) Polyphosphorylation negatively regulates Top1 activity. gTop1-13Myc purified from WT, vip1Δ, and vtc4Δ was incubated with supercoiled plasmid DNA to assess Top1 activity. Samples were spun, the supernatant was resolved on agarose gel and stained with ethidium bromide (top), and the myc beads were resolved by PAGE and immunoblotted. (B) Polyphosphorylation of K residues negatively regulates Top1 activity. GST-Top1 and GST-Top1 (K-L) were purified from WT, vip1Δ, and vtc4Δ. GST-Top1 activity was subsequently tested against supercoiled plasmid DNA as in (A). (C) Modulation of cellular polyP leads to regulation of Top1 polyphosphorylation status and activity. gTop1-13Myc WT yeast was phosphate starved for 2 hr, after which 10 mM of potassium phosphate was added and yeast grown for 2 hr. PolyP extracts were run on 30% PAGE and stained with toluidine blue (left), protein extracts were resolved and immunoblotted (top-right), and Top1 relaxation assay was performed as above (bottom-right). (D) Proposed functional model of Nsr1 and Top1 polyphosphorylation. Polyphosphorylation of K residues within the PASK domain of Top1 inactivates the protein in its capacity to relax supercoiled DNA and prevents its binding to Nsr1. Unmodified Top1 is functional and able to bind to Nsr1. Figures assembled from the same gel/membrane have stroked lines, and from different gel/membranes have solid lines. See also Figure S5. Molecular Cell 2015 58, 71-82DOI: (10.1016/j.molcel.2015.02.010) Copyright © 2015 Elsevier Inc. Terms and Conditions