PomX, a ParA/MinD ATPase activating protein, is a triple regulator of cell division in Myxococcus xanthus

Schumacher, Dominik; Harms, Andrea; Bergeler, Silke; Frey, Erwin; Søgaard‐Andersen, Lotte

doi:10.7554/elife.66160

Cited by 12 publications

(35 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Intriguingly, we see that the conserved basic residues in McdB contribute to the stability of this structure. For several other ParA/MinD-family ATPases, the associated partner proteins have also been shown, or suggested, to interact with their cognate ATPase via positively charged and disordered N-termini (57, 58, 59, 60, 61, 62). More recent data suggest the N-termini from some of these partner proteins can also fold and stabilize as α-helices upon interaction with their cognate ATPase (63, 64).…”

Section: Discussionmentioning

confidence: 99%

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

Basalla

Mak

Limcaoco

et al. 2022

Preprint

View full text Add to dashboard Cite

Carboxysomes are protein-based organelles essential for efficient CO2-fixation in cyanobacteria and some chemoautotrophic bacteria. We recently identified the two-component system responsible for spatially regulating carboxysomes, consisting of the proteins McdA and McdB. McdA is a member of the ParA/MinD-family of ATPases, which position a variety of cellular cargos across bacteria. McdB, however, represents a widespread but unstudied class of proteins. We previously found that McdB forms a hexamer and undergoes robust Liquid-Liquid Phase Separation (LLPS) in vitro, but the sequence and structural determinants underlying these properties are unknown. Here we define the domain architecture for McdB from the model cyanobacterium S. elongatus PCC 7942 which we use to dissect McdB oligomerization and LLPS. We identify an N-terminal Intrinsically Disordered Region (IDR), a central Q-rich dimerizing domain, and a C-terminal domain that trimerizes McdB dimers. Intriguingly, all three domains contributed to McdB LLPS. The Q-rich domain drove LLPS, the IDR tuned solubility, and the C-terminal domain provided further oligomerization to achieve full-length LLPS activity. We also identified critical basic residues in the IDR that modulate McdB LLPS, which we mutate to fine-tune condensate solubility both in vitro and in vivo. Our findings show that IDRs are not always drivers of LLPS, but can play secondary roles in modulating condensate solubility. Finally, we provide in silico evidence suggesting the N-terminal IDR of McdB acts as a MoRF, folding upon interaction with McdA. The data advance our understanding and application of carboxysomes, their positioning system, and the molecular grammar governing protein phase separation.SIGNIFICANCEThe recently characterized Maintenance of Carboxysome Distribution (Mcd) system is responsible for spatially regulating carbon-fixing organelles in bacteria called carboxysomes. Although an understanding of the Mcd system would advance our application of carboxysomes to help engineer carbon-fixing organisms and combat the climate crisis, one of its two essential components, McdB, has only recently been identified and is poorly understood. Here, we provide a thorough biochemical characterization of McdB from the model cyanobacterium S. elognatus. We define a structural model for McdB and identify how specific domains and residues contribute to its oligomerization and phase separation. Notably, we saw that a disordered region of McdB regulates phase separation in response to pH; impactful to both carboxysome regulation and protein phase separation.

show abstract

Section: Discussionmentioning

confidence: 99%

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

Basalla

Mak

Limcaoco

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Cargo specificity for A/D ATPases comes from interacting with a partner protein that either associates with the cargo, or is a physical component of the cargo itself. Partner proteins have a stretch of amino acids enriched in charged residues at the N-terminus that exclusively interacts with its A/D ATPase, while the rest of the partner protein is dedicated to cargo association (Schumacher et al, 2021) . We generated docking models of each A/D ATPase with an N-terminal peptide from its partner protein (Figure 7C) .…”

Section: Resultsmentioning

confidence: 99%

“…Despite their extreme diversity, data across the field supports the idea that partner proteins interact with and stimulate their A/D ATPases via a shared mechanism. Partner proteins involved in plasmid partition and chromosome segregation, as well as those required for positioning BMCs, flagella, chemotaxis clusters, and the divisome have all been shown, or suggested, to interact with their A/D ATPase via a positively charged and disordered N-terminus (Schumacher et al, 2021) . Our in silico analysis of A/D ATPase dimers docked with N-terminal peptides of their partner proteins demonstrates how specific cargos are assigned, and how these related positioning systems coexist and function in the same cell without cross-interference.…”

Section: Discussionmentioning

confidence: 99%

Multiple ParA/MinD ATPases coordinate the positioning of disparate cargos in a bacterial cell

Pulianmackal

Limcaoco

Ravi

et al. 2022

Preprint

View full text Add to dashboard Cite

SUMMARYIn eukaryotes, linear motor proteins govern intracellular transport and organization. In bacteria, where linear motors are absent, the ParA/MinD (A/D) family of ATPases spatially organize an array of genetic- and protein-based cellular cargos. ParA is well known to segregate plasmids and chromosomes, as is MinD for its role in divisome positioning. Less studied is the growing list of ParA/MinD-like ATPases found across prokaryotes and involved in the spatial organization of diverse protein-based organelles, such as Bacterial Microcompartments (BMCs), flagella, chemotaxis clusters, and conjugation machinery. Given the fundamental nature of these processes in both cell survival and pathogenesis, the positioning of these cargos has been independently investigated to varying degrees in several organisms. However, it remains unknown whether multiple A/D ATPases can coexist and coordinate the positioning of such a diverse set of fundamental cargos in the same cell. If so, what are the mechanistic commonalities, variation, and specificity determinants that govern the positioning reaction for each cargo? Here, we find that over a third of sequenced bacteria encode multiple A/D ATPases. Among these bacteria, we identified several human pathogens as well as the experimentally tractable organism, Halothiobacillus neapolitanus, which encodes seven A/D ATPases. We directly demonstrate that five of these A/D ATPases are each dedicated to the spatial regulation of a single cellular cargo: the chromosome, the divisome, the carboxysome BMC, the flagellum, and the chemotaxis cluster. We identify putative specificity determinants that allow each A/D ATPase to position its respective cargo. Finally, we show how the deletion of one A/D ATPase can have indirect effects on the inheritance of a cargo actively positioned by another A/D ATPase, stressing the importance of understanding how organelle trafficking, chromosome segregation, and cell division are coordinated in bacterial cells. Together, our data show how multiple A/D ATPases coexist and function to position a diverse set of fundamental cargos in the same bacterial cell. With this knowledge, we anticipate the design of minimal autonomous positioning systems for natural- and synthetic-cargos in bacteria for synthetic biology and biomedical applications.

show abstract

“…This architecture is conserved in PomX orthologs (Schumacher et al, 2021). PomX CC is required and sufficient for PomX polymerization and also interacts with PomY; both parts are required for PomX function (Schumacher et al, 2021). PomY comprises an N-terminal α-helical region predicted to form a coiled-coil (PomY CC ), followed by a region with six HEAT-repeats (PomY HEAT ), and a C-terminal mostly disordered region (PomY IDR ).…”

Section: Pomx Forms Filamentous Structures and Pomy Forms Biomolecula...mentioning

confidence: 99%

“…Upon division, the PomX/PomY/PomZ complex was suggested to undergo fission with both daughters "receiving" part of the complex (Schumacher et al, 2017). The three Pom proteins interact in all pairwise combinations and have distinct functions (Schumacher et al, 2017;Schumacher et al, 2021) While PomX/PomY/PomZ cluster translocation is well-understood based on experiments and theory (Bergeler and Frey, 2018;Kober et al, 2019;Schumacher et al, 2017) To understand the structure and function of the PomX/PomY/PomZ complex in vivo, we focused on PomX and PomY because they form a single cytoplasmic complex independently of PomZ that is able to stimulate Z-ring formation, while the PomZ ATPase is important for translocation of this complex (Schumacher et al, 2017). In strains expressing active mCherry (mCh) fusions, i.e.…”

Section: Introductionmentioning

confidence: 99%

A phase-separated biomolecular condensate nucleates polymerization of the tubulin homolog FtsZ to spatiotemporally regulate bacterial cell division

Ramm

Schumacher

Harms

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

SummaryCell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social, predatory bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single large megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we studied the structure and mechanism of this complex in vitro and in vivo. We demonstrate that PomY forms liquid-like biomolecular condensates by phase separation, while PomX self-assembles into filaments generating a single large cellular structure. The PomX structure enriches PomY, thereby guaranteeing the formation of precisely one PomY condensate per cell through surface-assisted condensation. In vitro, PomY condensates selectively enrich FtsZ and nucleate GTP-dependent FtsZ polymerization, suggesting a novel cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. PomY-nucleated FtsZ polymerization shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanism’s ancient origin.

show abstract

PomX, a ParA/MinD ATPase activating protein, is a triple regulator of cell division in Myxococcus xanthus

Cited by 12 publications

References 56 publications

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

Multiple ParA/MinD ATPases coordinate the positioning of disparate cargos in a bacterial cell

A phase-separated biomolecular condensate nucleates polymerization of the tubulin homolog FtsZ to spatiotemporally regulate bacterial cell division

Contact Info

Product

Resources

About