Fig 1.
90° Light Scattering (LS) assay profiles of MsFtsZ polymerisation.
Polymerisation in the presence of exogenous GTP or in the presence of exogenous GDP, ATP, and MsNDK or MtNDK (wild type or mutant). (A) Polymerisation of recombinant purified MsFtsZ. (B) Polymerisation of recombinant purified GDP-precharged MsFtsZ. The notations for the different samples, their positive and negative control samples are given in the inset text. GTP/ATP was added at the 200th sec, after developing a base line, to trigger polymerisation. The 40 sec delay in the polymerisation is indicated using a square bracket placed close to the X-axis and also depicted in the inset of the enlarged portion. Note the absence of the 40 sec delay in the case of the direct addition of GTP. The MsZ1mMGTP, MsZMsNdkWt1mMGDP1mMATP, and MsZMtNdkWt1mMGDP1mMATP reactions are common to A & B.
Fig 2.
SDS-PAGE profile of the FtsZ polymer pelleting assay for MsFtsZ polymerisation and its quantitation.
The SDS-PAGE profile of the sample in the pellet (A) and in the supernatant (B). Its quantitation is given as the pellet/supernatant ratio in (C). (A & B) Lanes: 1 & 9, MsFtsZ + GTP; 2 & 10, MsFtsZ without GTP; 3 & 11, MsFtsZ + MsNDK-Wt + GDP + ATP; 4 & 12, MsFtsZ + MsNDK-H117Q + GDP + ATP; 5 & 13, MsFtsZ + MtNDK-Wt + GDP + ATP; 6 & 14, MsFtsZ + MtNDK-H117Q + GDP + ATP; 7 & 15, GDP-precharged MsFtsZ + MsNDK-Wt + ATP; 8 & 16, GDP-precharged MsFtsZ + MtNDK-Wt + ATP. (C) Quantitations of the protein as the pellet/supernatant ratio in the reactions are represented by the bars, which are described in the figure itself. (D) The SDS-PAGE profile of the samples in the supernatant and pellet from MsFtsZ polymerisation in the absence of exogenous GDP. (E) Its quantitation given as the pellet/supernatant ratio. Lanes in (D): 1, MsFtsZ + GTP (Sup); 2, MsFtsZ—GTP (Sup); 3, MsFtsZ + MsNDK-Wt + ATP (Sup); 4, MsFtsZ + MsNDK-H117Q + ATP (Sup); 5, Marker; 6, MsFtsZ + GTP (Pellet); 7, MsFtsZ—GTP (Pellet); 8, MsFtsZ + MsNDK-Wt + ATP (Pellet); 9, MsFtsZ + MsNDK-H117Q + ATP (Pellet). (E) Quantitations of the protein as the pellet/supernatant ratio in the reactions are represented by the bars, which are described in the figure itself. (F-H) Comparison of TEM images (105k magnification) of FtsZ polymers formed from polymerisation of MsFtsZ triggered by MsNDKwt and MsNDKmut in the absence and presence of exogenous GDP. (F) MsFtsZ polymers from MsZ + NDKwt + ATP reaction; (G) MsFtsZ polymers from MsZ + NDKwt + GDP + ATP reaction; (H) MsFtsZ polymers from MsZ + NDKmut + ATP reaction. Images were captured in FEI Tecnai BioTWIN EM at 105k magnification.
Fig 3.
PEI-cellulose TLC profile of 32P-GTP formation during MsNDK-triggered MsFtsZ polymerisation and its quantitation.
(A) Formation of GTP analysed on TLC. (B) Quantitation of the GTP formed. (A) The TLC profile of 32P-GTP formation. Lanes: 1. γ32P-ATP alone; 2. α32P-GTP alone; 3. γ32P-ATP + MsNDK-Wt; 4. γ32P-ATP + MsNDK-H117Q; 5. γ32P-ATP + Purified MsFtsZ; 6. γ32P-ATP + Purified MsFtsZ + MsNDK-Wt; 7. γ32P-ATP + Purified MsFtsZ + MsNDK-H117Q; 8. γ32P-ATP + GDP-predepleted MsFtsZ + MsNDK-Wt; 9. γ32P-ATP + GDP + MsNDK-Wt; 10. γ32P-ATP + GDP + MsNDK-H117Q; 11. γ32P-ATP + GDP-precharged MsFtsZ; 12. γ32P-ATP + GDP-precharged MsFtsZ + MsNDK-Wt; 13. γ32P-ATP + GDP-precharged MsFtsZ + MsNDK-H117Q; 14. α32P-ATP + GDP-precharged MsFtsZ + MsNDK-Wt. (B). Quantitation of the 32P-GTP from the TLC profile. The sample names are described on the X-axis. The samples, corresponding to the TLC lanes 2 and 4, where 32P-GTP formation is not expected, are not shown in the bar graph quantitation profile. CMsZ stands for GDP-charged MsFtsZ and DMsZ stands for GDP-depleted MsFtsZ.
Fig 4.
Assay for the binding of γ32P-GTP to MsFtsZ (30 sec), SDS-PAGE profile of the UV-crosslinked γ32P-GTP-MsFtsZ, and the quantitation of the γ32P-GTP-MsFtsZ formed.
(A) SDS-PAGE profile of the UV-crosslinked γ32P-GTP-MsFtsZ bands and the autophosphorylated MsNDK monomer and dimer bands. Lanes: 1. GDP-precharged MsFtsZ + MsNDK-Wt + γ32P-ATP; 2. GDP-precharged MsZ + MsNDK-H117Q + γ32P-ATP; 3. GDP-precharged MsFtsZ + MtNDKWt + γ32P-ATP; 4. GDP-precharged MsFtsZ + MtNDK-H117Q + γ32P-ATP; 5. GDP-predepleted MsFtsZ + MsNDK-Wt + γ32P-ATP; see the autophosphorylated 32P-MsNDK dimer and monomer bands, due to the absence of GDP recipient substrate; 6. GDP-precharged MsFtsZ + MsNDK-Wt + α32P-ATP. (B) SDS-PAGE profile of the coomassie blue stained γ32P-GTP-MsFtsZ protein samples observed in (A). (C) Quantitation of the 32P-GTP-MsFtsZ formed from the UV-crosslinking. Left panel: phosphorimager profile of the UV-crosslinked 32P-GTP-MsFtsZ bands; Middle panel: the corresponding coomassie blue stained bands; Right panel: quantitation of the 32P-GTP-MsFtsZ. Lanes: 1. 32P-GTP-MsFtsZ formed from α32P-GTP and GDP-precharged MsFtsZ. 2. 32P-GTP-MsFtsZ formed from GDP-precharged MsFtsZ and γ32P-ATP, in the presence of MsNDK-wt. (D) PEI-cellulose TLC profile of γ32P-GTP formed. Lanes: 1. GDP-precharged MsFtsZ + MsNDK-Wt + γ32P-ATP; 2. GDP-precharged MsFtsZ + MsNDK-H117Q + γ32P-ATP; 3. GDP-precharged MsFtsZ + MtNDK-Wt + γ32P-ATP; 4. GDP-precharged MsFtsZ + MtNDK-H117Q + γ32P-ATP; 5. GDP-predepleted MsFtsZ + MsNDK-Wt + γ32P-ATP; 6. GDP-precharged MsFtsZ + MsNDK-Wt + α32P-ATP; 7. (E) Quantitation of the γ32P-GTP-MsFtsZ formed in the corresponding lanes 1–6 of panel A. cMsZ stands for GDP-charged MsFtsZ and dMsZ stands for GDP-depleted MsFtsZ.
Fig 5.
Assay for the formation of 32P-GTP from GDP bound to MsFtsZ and from free GDP.
(A) Left panel: 32P-GTP formation assay (30 sec) by PEI-cellulose TLC. Lanes: 1. GDP-precharged MsFtsZ + MsNDK-Wt + γ32P-ATP; 2. GDP-precharged MsFtsZ + MsNDKWt + γ32P-ATP + GDP; 3. GDP-predepleted MsFtsZ + MsNDK-Wt + γ32P-ATP; 4. GDP-predepleted MsFtsZ + MsNDK-Wt + γ32P-ATP + GDP. Right panel: Quantitation of the 32P-GTP spots in the TLC profile, using phosphorimager. Description of the samples is given within the diagram. (B) Phosphorimager profile of the UV-crosslinked, SDS-PAGE fractionated γ32P-GTP-FtsZ samples (left panel), Coomassie blue stained protein profile of the corresponding γ32P-GTP-FtsZ samples (middle panel), and the quantitation of the corresponding γ32P-GTP-FtsZ samples (right panel). Lanes: 1. GDP-precharged MsFtsZ + MsNDK-Wt + γ32P-ATP; 2. GDP-precharged MsFtsZ + MsNDKWt + γ32P-ATP + GDP; 3. GDP-predepleted MsFtsZ + MsNDK-Wt + γ32P-ATP; 4. GDP-predepleted MsFtsZ + MsNDK-Wt + γ32P-ATP + GDP. (C) Phosphorimager profile of the SDS-PAGE fractionated UV-crosslinked γ32P-GTP-FtsZ (left panel), the coomassie blue stained profile of the corresponding γ32P-GTP-FtsZ protein samples (middle panel), and the TLC profile of the assay for the γ32P-GTP formed in the corresponding samples (right panel) for MtFtsZ or MsFtsZ. Lanes: 1. GDP-precharged MtFtsZ + MtNDK-Wt + γ32P-ATP; 2. Denatured-refolded GDP-recharged MtFtsZ + MtNDK-Wt + γ32P-ATP; 3. GDP-precharged MsFtsZ + MsNDK-Wt + γ32P-ATP; 4. Denatured-refolded GDP-recharged MsFtsZ + MsNDK-Wt + γ-32P-ATP; 5. GDP-precharged MtFtsZ + MtNDK-Wt + γ32P-ATP + GDP; 6. Denatured-refolded GDP-recharged MtFtsZ + MtNDK-Wt + γ32P-ATP + GDP; 7. GDP-precharged MsFtsZ + MsNDK-Wt + γ32P-ATP + GDP; 8. Denatured-refolded GDP-recharged MsFtsZ + MsNDK-Wt + γ32P-ATP + GDP. All the 8 samples were analysed after the 30 sec reaction mentioned in the text. (D) UV-crosslinking assay. Lanes: 1. GDP-cross-linked MsFtsZ + MsNDK-Wt + γ32P-ATP; 2. MsFtsZ + α32P-GTP (cross-linked post-binding).
Fig 6.
Pull-down assays and co-immunoprecipitation showing FtsZ-NDK interaction.
(A) Pull-down assay using 6xHis-MtFtsZ and GST-MtNDK. Upper panel: GST-NDK is detected using anti-GST antibodies (mouse polyclonal). Lower panel: 6xHis-MtFtsZ is detected using anti-polyhistidine antibodies (mouse monoclonal). Lanes: 1. Ni2+-NTA agarose beads alone; 2. GST + 6xHis-MtFtsZ; 3. GST-MtNDK + 6xHis-MtFtsZ; 4. GST-MtNDK + 6xHis-MtFtsZ + anti-MtNDK antibodies (pre-incubated); 5. GST-MtNDK + 6xHis-MtFtsZ + anti-MtFtsZ antibodies (pre-incubated); 6. GST-MtNDK + 6xHis-MtFtsZ + anti-GST antibodies (pre-incubated). (B) Pull-down assay using 6xHis-EcFtsZ and GST-MtNDK. Upper panel: 6xHis-EcFtsZ is detected using anti-polyhistidine antibodies. Lower panel: GST-MtNDK is detected using anti-GST antibodies. Lanes: 1. GST-MtNDK + 6xHis-EcFtsZ; 2. GST-MtNDK + 6xHis-EcFtsZ + anti-MtNDK antibodies (pre-incubated); 3. GST-MtNDK + 6xHis-EcFtsZ + anti-EcFtsZ antibodies (pre-incubated); 4. GST-MtNDK + 6xHis-EcFtsZ + anti-GST antibodies (pre-incubated); 5. GST + 6xHis-EcFtsZ. Antibodies, whenever added to reaction mixtures were pre-incubated for 30 min at 4°C with the purified proteins at dilutions 1:100 for anti-MtNDK, anti-MtFtsZ, and anti-GST and 1:500 for anti-EcFtsZ before the addition of the cross-linker DTSP. The region where GST would have been on the blot is not shown. (C). Co-immunoprecipitation of recombinant MsNDK and MsFtsZ showing interaction. Lanes: 1–3. Co-IP with anti-MsNDK antibody; 4. Co-IP with anti-MsFtsZ antibody. Upper panel, western blotting with anti-MsFtsZ antibody; Lower panel, western blotting with anti-MsNDK antibody. Lanes: 1. MsNDK purified protein; 2. MsNDK, pre-incubated with anti-MsNDK antibody, followed by the addition of MsFtsZ; 3. MsNDK and MsFtsZ purified proteins; 4. MsFtsZ purified protein. (D) Co-immunoprecipitation of native MsNDK and MsFtsZ showing interaction in the total cell lysate. Lanes: 1. Purified recombinant 6x-His-MsNDK. 2. Protein A sepharose alone; 3. Protein A sepharose with M. smegmatis total cell lysate; 4. Co-IP with anti-MsNDK antibody; 5. Purified recombinant 6x-His-MsFtsZ; 6. Co-IP with anti-MsFtsZ antibody. Upper panel, western blotting with anti-MsFtsZ antibody; Lower panel, western blotting with anti-MsNDK antibody. Horizontal arrow heads indicates the endogenous MsNDK or MsFtsZ protein from the total cell lysate.