Mishra, Saurabh and Jakkala, Kishor and Srinivasan, Ramanujam and Arumugam, Muthu and Ranjeri, Raghavendra and Gupta, Prabuddha and Rajeswari, Haryadi and Ajitkumar, Parthasarathi (2015) NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK. In: PLOS ONE, 10 (12).
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Abstract
Introduction Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Methods Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90 degrees light scattering and FtsZ polymer pelleting assays. The gamma 32P-GTP synthesised by NDK from GDP and gamma 32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound P-32-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. Results NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. Conclusion Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.
| Item Type: | Journal Article |
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| Publication: | PLOS ONE |
| Publisher: | PUBLIC LIBRARY SCIENCE |
| Additional Information: | Copy right for this article belongs to the PUBLIC LIBRARY SCIENCE, 1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USA |
| Department/Centre: | Division of Biological Sciences > Microbiology & Cell Biology |
| Date Deposited: | 07 Jan 2016 07:12 |
| Last Modified: | 07 Jan 2016 07:12 |
| URI: | http://eprints.iisc.ac.in/id/eprint/53040 |
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