The Phosphatase PP2A Interacts With ArnA and ArnB to Regulate the Oligomeric State and the Stability of the ArnA/B Complex

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.

Keywords: Crenarchaea; archaellum; archaellum regulation; protein interaction; protein phosphatases; protein phosphorylation.

FIGURE 1

Expression of HA-tagged PP2A and PTP. (A) Growth curves of S. acidocaldarius cells grown in nutrient rich medium at 75°C were obtained for the parental MW001 strain (black solid circle) and the stains containing HA-tagged PP2A (black open square) and PTP (black open circle). The OD₆₀₀ of each strain was measured at the indicated time points. The average of three independent experiments is shown. (B) For motility assays, exponentially growing S. acidocaldarius cultures of around OD₆₀₀ 0.4 were spotted on semi-solid gelrite plates with 0.005% (w/v) NZ-amine and incubated at 75°C for 5 days before scanning the plates. The motility assays were repeated with three biological and six technical replicates. A representative experiment is shown. (C) Expression of HA-tagged PTP and (D) PP2A in S. acidocaldarius. PTPHA and PP2AHA mutants were grown in nutrient rich and starvation medium for 4 h. Samples were collected at different time points (0, 0.5, 1.0, 1.5, 2.0, and 4.0 h) and analyzed by Western blotting with α-HA. Representative Western blots are shown. A quantification of three independent experiments is shown below the blots.

FIGURE 2

Quantitative mass spectrometric analysis of PP2A and PTP affinity purifications (APs) from S. acidocaldarius. Individual proteins identified and quantified are displayed as circles in logarithmic plots of their molecular abundance (calculated as abundance_norm spec values, see Methods) versus their enrichment (ratio) in the target AP for (A) HA-PP2A and (B) HA-PTP relative to a control AP from wt cells. Circles in gray indicate non-specifically binding proteins, whereas the primary AP target proteins and specifically co-purified interactors are colored in red and light orange, respectively.

FIGURE 3

Reconstitution of the ArnA/B complex in vitro. (A) Purified Strep-ArnA (dashed line), ArnB-His (gray line), or 300 μg of ArnA-His and ArnB-Strep (black line) were incubated on ice for 1 h were loaded on a SD200 16/600 size exclusion column. Elution fractions were separated on SDS-PAGE and analyzed by Coomassie staining. The SDS-PAGE of the protein fractions eluting from the size exclusion column for the experiment where equal amounts ArnA-His and ArnB-Strep were mixed is shown. Mass photometry of (B) 100 nM ArnA, (C) 20 nM ArnB and (D) 50 nM ArnA/B complex.

FIGURE 4

In vitro interaction analysis assays of ArnA and ArnB. (A) ArnB-Strep (upper panel), ArnA-His and non-phos ArnB-Strep (left lanes of middle and lower panel) or ArnA-His and phos-ArnB-Strep (right lanes of middle and lower panel) were incubated at 55°C for 30 min and applied to a Ni-affinity resin, washed and eluted with imidazole. Fractions were analyzed by Western blotting with α-His (middle panel) and α-Strep antibodies (upper and lower panel). ST: starting materials; FT: flow-through; W1: 1st wash fraction; WL: last wash fraction; E: elution fraction. (B) ArnA/B complex collected from Figure 3A were incubated with Mn²⁺ and ATP in the presence (black line) and absence (gray line) of ArnC at 55°C for 30 min and were loaded on a Superdex 200 increase (10/300) size exclusion column. The upper panel depicts the elution pattern observed at 280 nm. Elution fractions were separated on SDS-PAGE and analyzed by Coomassie staining. The SDS-PAGEs of the protein fractions around the peaks observed are shown in the lower panel. (C) Mass photometry of the 50 nM ArnA/B complex.

FIGURE 5

Rates of phosphorylation of ArnB by ArnC and dephosphorylation by PP2A are increased in the ArnA/B complex. (A) 2 μM ArnA, ArnB, mixed ArnA/B mixture or isolated ArnA/B complex were incubated at 55°C in the presence of 100 μM ATP supplemented with [γ−³²P] ATP and 100 nM ArnC for different times, separated on SDS-PAGE and the phosphorylation levels were determined by phosphoimaging. (B) 2 μM ArnB, mixed ArnA/B or the isolated ArnA/B complex were first incubated at 55°C in the presence of 100 μM ATP supplemented with [γ−³²P] ATP and 100 nM ArnC. After 40 min, 100 fold excess of non-labeled ATP and 200 nM PP2A were added and samples were taken at different time points, separated on SDS-PAGE and the gels were analyzed by phosphoimaging (upper panel). (C) Quantification of phos ArnB signals from three independent dephosphorylation assays. Black circles, ArnB; empty squares, ArnA/B mixture; black filled triangles, ArnA/B complex.

FIGURE 6

PP2A interacts stronger with the phosphorylated ArnA/ArnB complex and dephosphorylation of the complex results in complex dissociation. (A) Strep-ArnA, (B) PP2A-his and non-phos/phos Strep-ArnA, (C) PP2A-his and non-phos/phos ArnB-Strep, and (D) PP2A-his and non-phos/phos Strep-ArnA/ArnB-Strep complex, were incubated at 55°C for 30 min and were applied to a Ni-affinity resin, and washed and eluted with imidazole. Fractions were analyzed by Western blotting with α-His (upper panel) and α-Strep antibodies (upper and lower panel). ST, starting materials; FT, flow-through; W1, 1st wash fraction; WL, last wash fraction; E, elution fraction.

FIGURE 7

Interaction with PP2A and dephosphorylation dissociates the ArnA/B complex. (A) Samples were incubated with Mn²⁺ at 55°C and were loaded on a Superdex 200 increase (10/300) size exclusion column. ArnB (blue line) or the ArnA/B complex (gray line) were incubated for 30 min. The ArnA/B complex was incubated for 30 min in the presence (orange line) or absence (black line) of ArnC and ATP followed by incubation with PP2A for 30 min. The ArnA/B complex incubated for 30 min with ArnC and ATP was indicated as a green line. The elution patterns observed at 280 nm are shown. (B) Elution fractions were separated on SDS-PAGE and analyzed by Coomassie staining. The SDS-PAGE of the protein fractions around the peaks observed are shown.

FIGURE 8

Binding of PP2A to the ArnA/B complex suffices to dissociate the complex. (A) PP2A (orange line) or the ArnA/B complex (gray line) were incubated for 30 min with EDTA at 55°C. The ArnA/B complex was incubated for 30 min in the presence of EDTA (black line) at 55°C followed by incubation with PP2A for 30 min at 55°C. Samples were loaded on a Superdex 200 increase (10/300) size exclusion column. The elution patterns observed at 280 nm are depicted. (B) Elution fractions were separated on SDS-PAGE and analyzed by Coomassie staining. The SDS-PAGE of the protein fractions around the peaks observed are shown.