Journal
MOLECULAR MICROBIOLOGY
Volume 116, Issue 3, Pages 943-956Publisher
WILEY
DOI: 10.1111/mmi.14781
Keywords
archaea; cell membrane structures; circadian clocks; locomotion; phosphorylation; single-molecule imaging
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Funding
- Deutsche Forschungsgemeinschaft [403222702, GSC-4]
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This study found that the ArlH protein in motile archaea has autophosphorylation activity and exists in a hexameric state when bound to hexameric ArlI. Mutagenesis of the putative catalytic residue in ArlH led to decreased autophosphorylation activity and impaired archaellation and motility in Sulfolobus acidocaldarius, indicating the crucial role of optimal phosphorylation activity of ArlH in archaellation and motility.
Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Since autophosphorylation and dephosphorylation of KaiC are central properties for the function of KaiC, we asked whether autophosphorylation is also a property of ArlH proteins. We observed that both ArlH from the euryarchaeon Pyrococcus furiosus (PfArlH) and from the crenarchaeon Sulfolobus acidocaldarius (SaArlH) have autophosphorylation activity. Using a combination of single-molecule fluorescence measurements and biochemical assays, we show that autophosphorylation of ArlH is closely linked to its oligomeric state when bound to hexameric ArlI. These experiments also strongly suggest that ArlH is a hexamer in its ArlI-bound state. Mutagenesis of the putative catalytic residue (Glu-57 in SaArlH) in ArlH results in a reduced autophosphorylation activity and abolished archaellation and motility in S. acidocaldarius, indicating that optimum phosphorylation activity of ArlH is essential for archaellation and motility.
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