Figure 4.
A dual-expression screen of Drosophila centrosome proteins identifies 6 soluble binary protein complexes. (A) Summary of screen and results. A total of 992 colonies were cultured and processed as described. (1a and 1b) The screen was conducted in two steps. Primary screening used Ni-NTA pulldowns followed by immuno-dot blots to detect His and GFP-tagged protein, resulting in 90 binary complexes. The secondary screen consisted of four steps: (2a) PCR was used to eliminate individual proteins that were dual tagged with both His and GFP and (2b) to remove duplicate plasmids. (2c) Cultures were scaled up for tandem pulldown/IP using first Ni-NTA and then GFP antibodies. Finally, (2d) the 21 positive interactions were verified with a reciprocal IP using anti-GFP. (B and B′) Six protein complexes were identified using the pExpress-Dual screen, which involved a tandem His6-pulldown/GFP-IP (B) and a final reciprocal GFP-IP step (B′). Immunoblots were probed with the indicated anti-His and GFP antibodies. The IP interaction numbers are the same as those in Fig. S2 B. Complexes 1, 9, and 43 were previously identified by other studies and serve as a strong proof of concept (Dzhindzhev et al., 2010; Slevin et al., 2012; McLamarrah et al., 2018). Interactions 18 and 45 are the subject of this study. Interaction 47 is a novel interaction. Note, G-box/PB1-PB2 binding is not preserved through the second GFP-IP step of the tandem pulldown/IP (interaction 18), demonstrating the relative low affinity of this interaction. (C) Left: Coomassie-stained SDS-PAGE showing purified MBP, MBP-Sas4 G-box, and partially purified His6-Plk4 PB1-PB2. Arrow indicates the position of His6-Plk4 PB1-PB2. Right: Immunoblot of the partially purified His6-Plk4 PB1-PB2 sample probed with anti-His6 antibody. (D) Binding isotherms derived from MST experiments. The His6-Plk4 PB1-PB2 concentration (50 mM) was constant in all experiments. The concentration of MBP and MBP-Sas4 was varied across more than four orders of magnitude (x axis). No binding of MBP to His6-PB1-PB2 was observed. Curve-fitting of the binding isotherm for MBP–G-box yielded a Kd of 28.2 µM (log10 concentration = −4.55 ± 0.14 M).

A dual-expression screen of Drosophila centrosome proteins identifies 6 soluble binary protein complexes. (A) Summary of screen and results. A total of 992 colonies were cultured and processed as described. (1a and 1b) The screen was conducted in two steps. Primary screening used Ni-NTA pulldowns followed by immuno-dot blots to detect His and GFP-tagged protein, resulting in 90 binary complexes. The secondary screen consisted of four steps: (2a) PCR was used to eliminate individual proteins that were dual tagged with both His and GFP and (2b) to remove duplicate plasmids. (2c) Cultures were scaled up for tandem pulldown/IP using first Ni-NTA and then GFP antibodies. Finally, (2d) the 21 positive interactions were verified with a reciprocal IP using anti-GFP. (B and B′) Six protein complexes were identified using the pExpress-Dual screen, which involved a tandem His6-pulldown/GFP-IP (B) and a final reciprocal GFP-IP step (B′). Immunoblots were probed with the indicated anti-His and GFP antibodies. The IP interaction numbers are the same as those in Fig. S2 B. Complexes 1, 9, and 43 were previously identified by other studies and serve as a strong proof of concept (Dzhindzhev et al., 2010; Slevin et al., 2012; McLamarrah et al., 2018). Interactions 18 and 45 are the subject of this study. Interaction 47 is a novel interaction. Note, G-box/PB1-PB2 binding is not preserved through the second GFP-IP step of the tandem pulldown/IP (interaction 18), demonstrating the relative low affinity of this interaction. (C) Left: Coomassie-stained SDS-PAGE showing purified MBP, MBP-Sas4 G-box, and partially purified His6-Plk4 PB1-PB2. Arrow indicates the position of His6-Plk4 PB1-PB2. Right: Immunoblot of the partially purified His6-Plk4 PB1-PB2 sample probed with anti-His6 antibody. (D) Binding isotherms derived from MST experiments. The His6-Plk4 PB1-PB2 concentration (50 mM) was constant in all experiments. The concentration of MBP and MBP-Sas4 was varied across more than four orders of magnitude (x axis). No binding of MBP to His6-PB1-PB2 was observed. Curve-fitting of the binding isotherm for MBP–G-box yielded a Kd of 28.2 µM (log10 concentration = −4.55 ± 0.14 M).

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