Competitive binding of MatP and topoisomerase IV to the MukB hinge domain
- London Institute of Medical Sciences, United Kingdom
- University of Oxford, United Kingdom
- Stanford University, United States
- University of Cambridge, United Kingdom
Abstract
Structural Maintenance of Chromosomes (SMC) complexes have ubiquitous roles in compacting DNA linearly, thereby promoting chromosome organization-segregation. Interaction between the Escherichia coli SMC complex, MukBEF, and matS-bound MatP in the chromosome replication termination region, ter, results in depletion of MukBEF from ter, a process essential for efficient daughter chromosome individualisation and for preferential association of MukBEF with the replication origin region. Chromosome-associated MukBEF complexes also interact with topoisomerase IV (ParC2E2), so that their chromosome distribution mirrors that of MukBEF. We demonstrate that MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding, which occurs with the same dimer to dimer stoichiometry. Furthermore, we show that matS DNA competes with the MukB hinge for MatP binding. Cells expressing MukBEF complexes that are mutated at the ParC/MatP binding interface are impaired in ParC binding and have a mild defect in MukBEF function. The data highlight competitive binding as a means of globally regulating MukBEF-topoisomerase IV activity in space and time.
Data availability
Source data files have been provided for all gel-based figures. Source data files for other assays have been included where indicated. All custom MATLAB scripts are provided as Source Code 1. All code used is previously published and also available via the cited reference(s). All reagents are available upon reasonable request.
Article and author information
Author details
Lidia K Arciszewska
Funding
Wellcome Trust (200782/Z/16/Z)
- David J Sherratt
Medical Research Council (MR/N020413/1)
- Carol V Robinson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Fisher et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Competitive binding of MatP and topoisomerase IV to the MukB hinge domain
eLife 10:e70444.
https://doi.org/10.7554/eLife.70444
Further reading
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Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.
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