Localized Dimerization and Nucleoid Binding Drive Gradient Formation by the Bacterial Cell Division Inhibitor MipZ

156

Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.

Section: Resultsmentioning

confidence: 52%

Section: Resultsmentioning

confidence: 75%

Bacterial scaffold directs pole-specific centromere segregation

Ptacin¹,

Gahlmann

Bowman³

et al. 2014

156

“…S3B), whereas our ATPase measurements used the same buffer as for protein targeting/translocation reactions (22). The transient nature of tetrameric Get3 could also render it susceptible to dissociation during size exclusion chromatography (23).…”

Section: Resultsmentioning

confidence: 99%

Precise timing of ATPase activation drives targeting of tail-anchored proteins

Rome

Rao

Clemons

et al. 2013

The localization of tail-anchored (TA) proteins, whose transmembrane domain resides at the extreme C terminus, presents major challenges to cellular protein targeting machineries. In eukaryotic cells, the highly conserved ATPase, guided entry of tail-anchored protein 3 (Get3), coordinates the delivery of TA proteins to the endoplasmic reticulum. How Get3 uses its ATPase cycle to drive this fundamental process remains unclear. Here, we establish a quantitative framework for the Get3 ATPase cycle and show that ATP specifically induces multiple conformational changes in Get3 that culminate in its ATPase activation through tetramerization. Further, upstream and downstream components actively regulate the Get3 ATPase cycle to ensure the precise timing of ATP hydrolysis in the pathway: the Get4/5 TA loading complex locks Get3 in the ATP-bound state and primes it for TA protein capture, whereas the TA substrate induces tetramerization of Get3 and activates its ATPase reaction 100-fold. Our results establish a precise model for how Get3 harnesses the energy from ATP to drive the membrane localization of TA proteins and illustrate how dimerization-activated nucleotide hydrolases regulate diverse cellular processes.

“…Processes regulated by ParA-like proteins often are regulated by or are dependent on associated proteins that modulate ParA ATPase activity (17,18,26,(28)(29)(30)(31). It is possible that ParP similarly modulates ParC's enzymatic activity; to date, we have been unable to purify ParP to test this possibility.…”

Section: Discussionmentioning

confidence: 99%

ParP prevents dissociation of CheA from chemotactic signaling arrays and tethers them to a polar anchor

Ringgaard

Zepeda-Rivera

et al. 2013

Significance Targeting of cellular components to a particular site in a cell is often a highly regulated process, even in cells as small as bacteria. Robust chemotactic signaling, which is used by motile bacteria to survey their environments and navigate in response to them, requires appropriate cellular distribution of a large chemosensory apparatus. Here, we report how polarly flagellated vibrios ensure polar localization of their chemotactic machinery by capturing signaling proteins at the pole. Polar localization is mediated by a tripartite protein interaction network in which one protein prevents disassociation of a key signaling component from chemotactic complexes and tethers the complexes to a polar anchor. Polar tethering and localization are prerequisites for proper chemotaxis.