Hsp90 of E. coli modulates assembly of FtsZ, the tubulin homolog in E. coli
Thursday, September 14, 2017 — Poster Session III
- A Balasubramanian
- M Markovski
- S Wickner
Heat shock protein 90 (Hsp90) is a highly conserved ATP dependent molecular chaperone involved in remodeling, activating and stabilizing numerous client proteins. Since many Hsp90 client proteins have been linked to cancer and other diseases, understanding the functions of Hsp90 is important. The Hsp90 homolog in E. coli, Hsp90Ec, has been shown to cause cell filamentation when overexpressed. By observing the cells under light microscopy, we observed that the filamentous cells had distinct nucleoids, indicating that Hsp90Ec overexpression does not affect chromosomal replication or segregation. To assess if overexpression of Hsp90Ec interferes with the cell division machinery, we tested if FtsZ, a tubulin homolog essential for cell division, assembled into ring-like structures at future sites of cell division as it does in cells not overexpressing Hsp90Ec. We observed by immunofluorescence of fixed cells that FtsZ rings were not detectable in Hsp90Ec overexpressing cells. We also found that FtsZ was present at normal levels in cells overexpressing Hsp90Ec. Together these results suggest high levels of Hsp90Ec affect FtsZ assembly. To test if the Hsp90Ec stabilized negative regulators of FtsZ, we singly deleted genes coding for negative regulators SulA, ClpX, MinC and SlmA but none of these mutants reversed the filamentous phenotype seen in Hsp90Ec overexpressing cells. We next tested the hypothesis that Hsp90Ec prevents FtsZ polymerization. Using purified proteins and fluorescence microcopy, we observed that fluorescently labeled FtsZ formed filaments and bundles in the absence of Hsp90Ec, but not in the presence. Additionally, we showed that light scattering by FtsZ polymers was inhibited when Hsp90Ec was added prior to polymerization. We further observed that an ATP hydrolysis defective Hsp90Ec mutant retained ability to inhibit FtsZ polymerization, consistent with the known ability of Hsp90 to interact with clients independent of ATP hydrolysis. Moreover, we observed that Hsp90Ec client-binding defective mutants exhibited reduced ability to prevent FtsZ polymerization in vitro. In summary, our data show that Hsp90Ec, when overexpressed, inhibits divisome assembly in vivo and prevents FtsZ polymerization in vitro. They suggest that Hsp90Ec may modulate of cell division by interacting and holding FtsZ, possibly slowing cell division during heat stress and other stresses.
Category: Molecular Biology and Biochemistry