Abstract

In the crenarchaeon Sulfolobus acidocaldarius, the archaellum, a type-IV pilus like motility structure, is synthesized in response to nutrient starvation. Synthesis of components of the archaellum is controlled by the archaellum regulatory network (arn). Protein phosphorylation plays an important role in this regulatory network since the deletion of several genes encoding protein kinases and the phosphatase PP2A affected cell motility. Several proteins in the archaellum regulatory network can be phosphorylated, however, details of how phosphorylation levels of different components affect archaellum synthesis are still unknown. To identify proteins interacting with the S. acidocaldarius phosphatases PTP and PP2A, co-immunoprecipitation assays coupled to mass spectrometry analysis were performed. Thirty minutes after growth in nutrient starvation medium, especially a conserved putative ATP/GTP binding protein (Saci_1281), a universal stress protein (Saci_0887) and the archaellum regulators ArnA and ArnB were identified as highly abundant interaction proteins of PP2A. The interaction between ArnA, ArnB, and PP2A was further studied. Previous studies showed that the Forkhead-associated domain containing ArnA interacts with von Willebrand type A domain containing ArnB, and that both proteins could be phosphorylated by the kinase ArnC in vitro. The ArnA/B heterodimer was reconstituted from the purified proteins. In complex with ArnA, phosphorylation of ArnB by the ArnC kinase was strongly stimulated and resulted in formation of (ArnA/B)2 and higher oligomeric complexes, while association and dephosphorylation by PP2A resulted in dissociation of these ArnA/B complexes.

Highlights

  • Protein phosphorylation is one of the most important post-translational modifications in the three domains of life (Mok et al, 2010; Pereira et al, 2011; Reimann et al, 2013)

  • Cultivation of S. solfataricus cells with different carbon sources caused a significant change of phospho-protein patterns, especially proteins involved in central carbohydrate metabolism, which suggested a role of protein phosphorylation in regulating the carbon flux

  • PTP and PP2A are the only two protein phosphatases in S. acidocaldarius. Deletion mutants of these phosphatases show higher phosphorylation levels of several proteins in the archaellum regulatory network and deletion of PP2A resulted in increased expression of the archaellum and increased motility (Reimann et al, 2013)

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Summary

Introduction

Protein phosphorylation is one of the most important post-translational modifications in the three domains of life (Mok et al, 2010; Pereira et al, 2011; Reimann et al, 2013). Large-scale phosphoproteomic analysis is a widely used approach to understand the role of protein phosphorylation in different cellular processes. In the Crenarchaeon S. solfataricus P2, 540 unique Ser/Thr/Tyr-phosphoproteins were likewise identified by phosphoproteomic analysis, suggesting an important regulatory role of protein phosphorylation in S. solfataricus (Esser et al, 2012). Cultivation of S. solfataricus cells with different carbon sources (glucose and tryptone) caused a significant change of phospho-protein patterns, especially proteins involved in central carbohydrate metabolism, which suggested a role of protein phosphorylation in regulating the carbon flux. The ratio of pTyr to pSer/pThr posttranslational modification was much higher in S. solfataricus and S. acidocaldarius than in Bacteria and Eukarya, which suggested tyrosine phosphorylation plays a much more important role in Sulfolobales, possibly to complement the absence of TCS

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