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bioRxiv · 10.1101/2024.02.14.580282

Differential GTP-dependent in-vitro polymerization of recombinant Physcomitrella FtsZ proteins

Milferstaedt, S. W. L.·Joest, M.·Hoernstein, S. N. W.·Bohlender, L. L.·Oezdemir, B.·van der Does, C.·Decker, E. L.·Reski, R.
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Abstract

1.Bacterial cell division and plant chloroplast division require self-assembling Filamentous temperature-sensitive Z (FtsZ) proteins. FtsZ proteins are GTPases sharing structural and biochemical similarities with eukaryotic tubulin. In the moss Physcomitrella, the morphology of the FtsZ polymer networks varies between the different FtsZ isoforms. The underlying mechanism and foundation of the distinct networks is unknown. Here, we investigated the interaction of Physcomitrella FtsZ2-1 with FtsZ1 isoforms via co-immunoprecipitation and mass spectrometry, and found protein-protein interaction in vivo. We tagged FtsZ1-2 and FtsZ2-1 with different fluorophores and expressed both in E. coli, which led to the formation of defined structures within the cells and to an influence on bacterial cell division and morphology. Furthermore, we have optimized the purification protocols for FtsZ1-2 and FtsZ2-1 expressed in E. coli and characterized their GTPase activity and polymerization in vitro. Both FtsZ isoforms showed GTPase activity. Stoichiometric mixing of both proteins led to a significantly increased GTPase activity, indicating a synergistic interaction between them. In light scattering assays, we observed GTP-dependent assembly of FtsZ1-2 and of FtsZ2-1 in a protein concentration dependent manner. Stoichiometric mixing of both proteins resulted in significantly faster polymerization, again indicating a synergistic interaction between them. Under the same conditions used for GTPase and light scattering assays both FtsZ isoforms formed filaments in a GTP-dependent manner as visualized by transmission electron microscopy (TEM). Taken together, our results reveal that Physcomitrella FtsZ1-2 and FtsZ2-1 are functionally different, can synergistically interact in vivo and in vitro, and differ in their properties from FtsZ proteins from bacteria, archaea and vascular plants.

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Milferstaedt, S. W. L., Joest, M., Hoernstein, S. N. W., Bohlender, L. L., Oezdemir, B., van der Does, C., Decker, E. L., Reski, R.. 2024-02-15. Differential GTP-dependent in-vitro polymerization of recombinant Physcomitrella FtsZ proteins. https://doi.org/10.1101/2024.02.14.580282

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