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. 2018 Dec 19;87(1):e00523-18. doi: 10.1128/IAI.00523-18

FIG 1.

FIG 1

Recombinant PfPKG interacts with RPT1 and HSP90 in vitro. A split-luciferase protein complementation assay was used to test interaction between the PfPKG kinase domain (PfPKG E706A and PfPKG R781A) and a fragment of RPT1 (amino acids 114 to 420) or HSP90 (amino acids 228 to 518). The proteins were expressed in wheat germ extracts as fusions with the N-terminal domain (Nfluc) or the C-terminal domain (Cfluc) of luciferase. Reconstitution of functional luciferase was measured as average relative light units (RLU). (A) Western blotting confirmed expression of fusion proteins in in vitro-transcribed/translated wheat germ extracts. These fusion proteins were used in subsequent complementation assays. Fusions with Nfluc were detected using antiserum against the N-terminal domain of firefly luciferase. Fusions with Cfluc were detected using anti-FLAG antiserum. Smaller antibody-reactive bands are the likely translation products of incomplete transcripts. Expected molecular weights were as follows: Nfluc-PfPKG E706A, ∼100 kDa; Nfluc-HSP90228–518, ∼85 kDa; Nfluc-RPT1114–420, ∼85 kDa; Cfluc-PfPKG E706A, ∼85 kDa; Cfluc-PfPKG R781A, ∼85 kDa; Cfluc-HSP90228–518, ∼70 kDa; Cfluc-RPT1114–420, ∼60 kDa. (B) Incubation of Nfluc-PfPKG E706A with Cfluc-RPT1114–420, Cfluc-PfPKG E706A with Nfluc-RPT1114–420, or Cfluc-PfPKG R781A with Nfluc-RPT1114–420 significantly increased luciferase activity. In contrast, incubation of Nfluc-PfPKG E706A with Cfluc without a fusion partner [Cfluc(-)], Cfluc-PfPKG E706A with Nfluc without a fusion partner [Nfluc(-)], and Cfluc-RPT1114–420 with Nfluc(-) or Cfluc-PfPKG R781A with Nfluc(-) had a minimal effect on luciferase activity. Results shown are average of three replicates ± standard deviations. (C) Incubation of Nfluc-PfPKG E706A with Cfluc-HSP90228–518, Cfluc-PfPKG E706A with Nfluc-HSP90228–518, or Cfluc-PfPKG R781A with Nfluc-HSP90228–518 significantly increased luciferase activity. In contrast, incubation of Nfluc-PfPKGE706A with Cfluc(-), Cfluc-PfPKGE706A with Nfluc(-), Cfluc-RPT1114–420 with Nfluc(-), or Cfluc-PfPKG R781A with Nfluc(-) had a minimal effect on luciferase activity. Results shown are average of three replicates ± standard deviations. Data were analyzed using an unpaired t test (*, P < 0.05; **, P < 0.005).