Journal: The Journal of Biological Chemistry

Article Title: Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

doi: 10.1074/jbc.RA119.011680

Figure Lengend Snippet: Polyphosphorylation can be exacerbated during the extraction procedures. A, cultures from DDY1810 yeast containing different levels of polyP (no polyP- vtc4 Δ, normal polyP-WT, and high polyP- vip1 Δ) were mixed in a 1:1 ratio (OD 600 = 0.8) with a culture from gTop1–13Myc- or gNsr1–13Myc-tagged vtc4 Δ; the proteins were extracted, run on NuPAGE, and blotted with anti-Myc to detect Top1 and Nsr1 and with anti-tubulin (α- Tub ) (loading control). B, total polyP from the ppn1 Δ ppn2 Δ ppx1 Δ ddp1 Δ quadruple DDY1810 mutant (DQM). PolyP corresponding to 5 μg of total RNA was fractionated on a 30% polyacrylamide gel and stained by negative staining with DAPI. C, shift-up experiment of purified gNsr1–13Myc ( vtc4 Δ), kept on Myc-agarose beads, subjected to several treatments, in short, the beads were incubated for 20 min at 30 °C either with 2 m m polyP100 ( P100 ), polyP45 ( P45 ), polyP extracted from the ppn1 Δ ppn2 Δ ppx1 Δ ddp1 Δ DDY1810 quadruple mutant ( DQM ) or left untreated, washed in lysis buffer, and further incubated with polyP as described in panel C . Protein samples were run on NuPAGE and blotted with anti-Myc. The figures presented are a representation of at least three independent repeats.

Article Snippet: Yeast strains were grown in SC media: 20% glucose, yeast nitrogen base without amino acids (Sigma, Y0626) complemented with complete synthetic media (CM: MPBio-4500-022) if WT; complete synthetic dropout media without uracil (CSM-URA: MPBio-4511-222) if transformed with GPD-vt-Ppx1 vector or pCA384 (GFP–Top1); or complete synthetic dropout media without tryptophan (CSM-TRP: MPBio-4511-022) if transformed with pCA511 (GFP–Top1(D/E-A/L)).

Techniques: Mutagenesis, Staining, Negative Staining, Purification, Incubation, Lysis

Journal: The Journal of Biological Chemistry

Article Title: Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

doi: 10.1074/jbc.RA119.011680

Figure Lengend Snippet: Polyphosphorylation effect on Top1 and Nsr1 mobility shift. A, schematic representation of the putative models for K-PPn mobility shift enhancement. The replacement model suggests that the polyP in a K-PPn target protein can be substituted on the same lysine residue by the additional polyP. The additional model suggests that polyphosphorylation, exposes buried lysine residues, can then be de novo polyphosphorylated once further polyP is available. B, testing the replacement model. Shift-up experiment of purified unpolyphosphorylated gTop1–13Myc ( vtc4 Δ) was kept on Myc-agarose beads and subjected to several treatments; in short, the beads were first incubated for 20 min at 30 °C either with 2 m m P45 or 0.5 m m bis-FAM-P8 or left untreated, washed in lysis buffer (see under “Experimental procedures”), and then further incubated as described in the figure. Protein samples were run on NuPAGE and blotted with anti-FITC to detect transfer of polyP from bis-FAM-P8 to Top1 and with anti-Myc to detect Top1 mobility shift. C, shift-up experiment of total protein from gTop1–13Myc–tagged vtc4 Δ yeast mutant extracted under denaturing conditions (Lysis Buffer (LB) with 2% SDS; see under “Experimental procedures”) or under normal conditions (LB), incubated for 20 min at 30 °C with either 2 m m P65 or P100 or left untreated, run on NuPAGE, and blotted with anti-Myc (to detect Top1), anti-Nsr1, and anti-tubulin (as loading control). D, shift-up experiment of total protein from gTop1–13Myc-tagged vtc4 Δ yeast mutant extracted under native conditions and split in two; one part was denatured by adding SDS to 2% and the other part was kept under native conditions. Protein samples were subsequently treated with increasing concentrations of P100 for 10 min at 30 °C, run on NuPAGE, and blotted with anti-Myc (to detect Top1), anti-Nsr1, and anti-tubulin (α- Tub ) (as loading control).

Article Snippet: Yeast strains were grown in SC media: 20% glucose, yeast nitrogen base without amino acids (Sigma, Y0626) complemented with complete synthetic media (CM: MPBio-4500-022) if WT; complete synthetic dropout media without uracil (CSM-URA: MPBio-4511-222) if transformed with GPD-vt-Ppx1 vector or pCA384 (GFP–Top1); or complete synthetic dropout media without tryptophan (CSM-TRP: MPBio-4511-022) if transformed with pCA511 (GFP–Top1(D/E-A/L)).

Techniques: Mobility Shift, Purification, Incubation, Lysis, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

doi: 10.1074/jbc.RA119.011680

Figure Lengend Snippet: Glutamic and aspartic acids play an essential role in Top1 polyphosphorylation. A , nuclear distribution of WT yeast exogenously expressing GFP–Top1 and GFP–Top1(D/E-A/L) by confocal microscopy. DIC , differential interference contrast. Scale bar , 5 μm. B, GFP–Top1 and GFP–Top1(D/E-A/L) exogenously expressed in WT yeast were extracted, run on NuPAGE and blotted with anti-GFP and anti-Tubulin (α- Tub ) (loading control). C, GFP–Top1 and GFP–Top1(D/E-A/L) expression levels were measured by FACS. The mean fluorescence intensity of the distribution is given ( n = 3). All yeast strains are in DDY1810 background. The figures presented are a representation of at least three independent repeats.

Article Snippet: Yeast strains were grown in SC media: 20% glucose, yeast nitrogen base without amino acids (Sigma, Y0626) complemented with complete synthetic media (CM: MPBio-4500-022) if WT; complete synthetic dropout media without uracil (CSM-URA: MPBio-4511-222) if transformed with GPD-vt-Ppx1 vector or pCA384 (GFP–Top1); or complete synthetic dropout media without tryptophan (CSM-TRP: MPBio-4511-022) if transformed with pCA511 (GFP–Top1(D/E-A/L)).

Techniques: Expressing, Confocal Microscopy, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

doi: 10.1074/jbc.RA119.011680

Figure Lengend Snippet: Effect of phosphatases on polyphosphate and on polyphosphorylation target mobility. A, yeast protein extracts from the strains indicated in the figure were extracted in native buffer (LB+ 2% SDS), run on NuPAGE, and blotted with anti-Nsr1. B, total polyP from gTop1–9Myc-tagged ppn1 Δ ppn2 Δ ppx1 Δ ddp1 Δ BY4741 quadruple mutant ( BQM ). PolyP corresponding to 5 μg of total RNA was fractionated on a 30% polyacrylamide gel, stained by negative staining with DAPI ( left panel ), and quantified by malachite green ( n = 4, right panel ). n/s , not significant. C, gTop1–9Myc endogenously tagged in BY4741 WT ( BWT ) or ppn1 Δ ppn2 Δ ppx1 Δ ddp1 Δ BY4741 quadruple mutant ( BQM ) backgrounds was extracted under the conditions described in the figure, run on NuPAGE, and blotted with anti-Myc (to detect Top1), anti-Nsr1, and anti-tubulin (α- Tub ) (as a loading control). All yeast strains are in BY4741 background unless otherwise stated. The figures presented are a representation of at least three independent repeats.

Article Snippet: Yeast strains were grown in SC media: 20% glucose, yeast nitrogen base without amino acids (Sigma, Y0626) complemented with complete synthetic media (CM: MPBio-4500-022) if WT; complete synthetic dropout media without uracil (CSM-URA: MPBio-4511-222) if transformed with GPD-vt-Ppx1 vector or pCA384 (GFP–Top1); or complete synthetic dropout media without tryptophan (CSM-TRP: MPBio-4511-022) if transformed with pCA511 (GFP–Top1(D/E-A/L)).

Techniques: Mutagenesis, Staining, Negative Staining

Journal: The Journal of Biological Chemistry

Article Title: Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

doi: 10.1074/jbc.RA119.011680

Figure Lengend Snippet: Top1 and Nsr1 mobility in different BY4741 mutants. A, effect of deleting all known polyphosphatases on Nsr1 mobility. Proteins from different mutants were extracted under native (LB; top panel ) or denaturing conditions (LB + 2% SDS; bottom panel ), run on NuPAGE, and blotted with anti-Nsr1. B, effect of deleting all known polyphosphatases on Top1 mobility. Proteins from different mutants tagged with gTop1–9Myc were processed as in A and blotted with anti-Myc. All yeast strains are in the BY4741 background. The figures presented are a representation of at least three independent repeats.

Article Snippet: Yeast strains were grown in SC media: 20% glucose, yeast nitrogen base without amino acids (Sigma, Y0626) complemented with complete synthetic media (CM: MPBio-4500-022) if WT; complete synthetic dropout media without uracil (CSM-URA: MPBio-4511-222) if transformed with GPD-vt-Ppx1 vector or pCA384 (GFP–Top1); or complete synthetic dropout media without tryptophan (CSM-TRP: MPBio-4511-022) if transformed with pCA511 (GFP–Top1(D/E-A/L)).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

doi: 10.1074/jbc.RA119.011680

Figure Lengend Snippet: Engineering a yeast strain to study endogenous polyphosphorylation in DDY1810 WT background. A, schematic representation of WT yeast cells expressing a vacuolar ( vt ) localized Ppx1 ( vt-Ppx1 ; bottom panel ) or an empty vector ( top panel ). The presence of polyP in the vacuole is shown in dark gray as seen by EM . B, total polyP from gTop1–13Myc-tagged WT cells overexpressing empty vector control or vt-Ppx1. PolyP corresponding to 5 μg of total RNA was fractionated on a 30% polyacrylamide gel, stained by negative staining with DAPI ( left ), and quantified by malachite green assay ( right , n = 4). C, nuclear polyP of the same strains as in A . Nuclear polyP corresponding to 30 μg of total RNA was run as in B ( right, n = 4 (two biological replicates and two technical replicates)). D , localization of vt-Ppx1 by immunofluorescence. The vt-Ppx1 is stained in green and the nucleus in blue ( V, vacuole; N, nucleus; PN, perinuclear; CER, cortical endoplasmic reticulum; i scale bar, 30 μm; ii scale bar, 3 μm iii scale bar, 1.5 μm. E, comparison of Top1 mobility in gTop1–13Myc-tagged vtc4 Δ and in WT yeast overexpressing empty vector or vt-Ppx1. F, comparison of Nsr1 mobility in vtc4 Δ and in WT yeast overexpressing empty vector or vt-Ppx1. The figures presented are a representation of at least three independent repeats unless otherwise stated. * represents Nsr1 real mobility shift under polyphosphorylation; ** represents Nsr1 mobility shift upon contact with the vacuolar localized polyP.

Article Snippet: Yeast strains were grown in SC media: 20% glucose, yeast nitrogen base without amino acids (Sigma, Y0626) complemented with complete synthetic media (CM: MPBio-4500-022) if WT; complete synthetic dropout media without uracil (CSM-URA: MPBio-4511-222) if transformed with GPD-vt-Ppx1 vector or pCA384 (GFP–Top1); or complete synthetic dropout media without tryptophan (CSM-TRP: MPBio-4511-022) if transformed with pCA511 (GFP–Top1(D/E-A/L)).

Techniques: Expressing, Plasmid Preparation, Staining, Negative Staining, Malachite Green Assay, Immunofluorescence, Mobility Shift

Journal: The Journal of Biological Chemistry

Article Title: Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

doi: 10.1074/jbc.RA119.011680

Figure Lengend Snippet: Engineering a yeast strain to study endogenous polyphosphorylation in DDY1810 ppn1 Δ ppn2 Δ background. A, total polyP from gTop1–3HA-tagged ppn1 Δ ppn2 Δ cells overexpressing empty vector control or vt-Ppx1 and gTop1–3HA-tagged vtc4 Δ overexpressing empty vector control. PolyP corresponding to 5 μg of total RNA was fractionated on a 30% polyacrylamide gel, stained by negative staining with DAPI ( left ), and quantified by malachite green assay ( n = 4). B, nuclear polyP of the same strains as in A . PolyP was run as in A but with polyP corresponding to 40 μg of RNA ( n = 3 (two biological replicates in which one of the biological replicates has two technical replicates)). C, comparison of Top1 and Nsr1 mobility in total protein extracts ( left panel ) and nuclear protein extracts ( right panel , n = 2) from gTop1–3HA-tagged vtc4 Δ and ppn1 Δ ppn2 Δ ( DDM ) overexpressing vt-Ppx1 or empty vector. Protein samples were extracted under denaturing conditions, run on NuPAGE, and blotted with anti-HA (to detect Top1), anti-Nsr1, anti-VPH1 (as a vacuolar marker control), anti-Ppx1, and anti-histone 3 (as a nuclear marker control). All yeast strains are in a DDY1810 background. The figures presented are a representation of at least three independent repeats unless otherwise stated.

Article Snippet: Yeast strains were grown in SC media: 20% glucose, yeast nitrogen base without amino acids (Sigma, Y0626) complemented with complete synthetic media (CM: MPBio-4500-022) if WT; complete synthetic dropout media without uracil (CSM-URA: MPBio-4511-222) if transformed with GPD-vt-Ppx1 vector or pCA384 (GFP–Top1); or complete synthetic dropout media without tryptophan (CSM-TRP: MPBio-4511-022) if transformed with pCA511 (GFP–Top1(D/E-A/L)).

Techniques: Plasmid Preparation, Staining, Negative Staining, Malachite Green Assay, Marker