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Mapping of the differential exchange & dynamics of type IVa pilus machine components: Implications for function
Large multiprotein complexes perform various biological processes. In the past decade, in vivo fluorescence labeling of proteins and advanced microscopy techniques have revealed that many protein complexes, previously assumed to be static, contain components that dynamically exchange subunits with c...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2023
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| Zusammenfassung: | Large multiprotein complexes perform various biological processes. In the past decade, in vivo fluorescence labeling of proteins and advanced microscopy techniques have revealed that many protein complexes, previously assumed to be static, contain components that dynamically exchange subunits with cellular pools. The type IVa pilus machine (T4aPM) drives the extension/adhesion/retraction cycles of T4aP and is the prototype of a large family of prokaryotic cell envelope-spanning macromolecular complexes involved in motility, DNA-uptake, secretion, and adhesion. Here, we used the T4aPM in Myxococcus xanthus as a model to investigate whether proteins of this nano-machine dynamically exchange and if exchange is coupled to the activity of the T4aPM. We performed Fluorescence Recovery After Photobleaching (FRAP) experiments on live cells, in which selected fluorescently-tagged proteins of the T4aPM were synthesized at close to native levels. We demonstrate different dynamics for individual components of the T4aPM. The PilQ secretin and its associated periplasmic protein TsaP, that jointly form the outer membrane (OM) pore, as well as the inner membrane (IM) proteins PilP and PilO, and likely also the IM protein PilN, that jointly form the alignment complex connecting the outer OM pore to the IM platform protein PilC, were stably incorporated into the T4aPM. By contrast, the three cytoplasmic proteins PilM, PilB and PilT were dynamically incorporated into the T4aPM and displayed different exchange kinetics. In the case of PilM, the exchange rate was dependent on the functional state of the T4aPM and were reduced by binding of the extension and retraction ATPases, PilB and PilT, to the base of the T4aPM. Remarkably, using an active variant of the PilM that was stably incorporated into the T4aPM, we demonstrate that PilM exchange is not essential for T4aPM function. The ATPases PilB and PilT underwent rapid exchange; however, PilB exchange was much faster than PilT exchange, supporting that the rate of T4aP formation is limited by slow PilT unbinding from the T4aPM. The different exchange kinetics of PilM, PilB and PilT support that they exchange independently of each other. Moreover, our data support that the dynamic exchange of PilB and PilT reflect neither the incorporation of the hundreds of individual PilA building block of the T4aP per second into the extending pilus nor their removal during retractions, and that the PilA subunits are delivered and removed to the T4aPM independently of any T4aPM component. We conclude that the trans-envelope structural elements of the T4aPM are stable structures once assembled, that the only dynamically incorporated proteins are the cytoplasmic PilM, PilB and PilT, with PilM exchange not being important for T4aPM function, while PilB and PilT exchange reflects the binding and unbinding of these proteins to the T4aPM to power T4aP extension and retraction, respectively.
The second part of this study focus on SgmO, a regulator of exopolysaccharide synthesis. Detailed genetic analyses confirmed that SgmO is important for exopolysaccharide synthesis, T4aP-dependent motility, and development. Moreover, ΔsgmO cells had reduced T4aP levels. |
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| DOI: | 10.17192/z2024.0097 |