The C Terminus of Alb3 Interacts with the Chromodomains 2 and 3 of cpSRP43

Lewis et al. (1) studied the interaction between cpSRP43 and Alb3 as we did previously (2), but they come to different results, on which we would like to comment.

Using isothermal titration calorimetry (ITC) they report a K_d for the complex of cpSPR43 and the C terminus of Alb3 (A3CT), which is 100-fold lower than that observed by us and conclude that the differences in the K_d values are due to the presence of 5% (w/v) glycerol in our experiments. However, glycerol is a commonly used additive in ITC measurements, and concentrations up to 30% have not been reported to cause artifacts (3). Our control experiments also did not provide any evidence for this assumption (Fig. 1A). In the absence of glycerol, a K_d of 13.2 μm for the cpSRP43-A3CT complex was observed (Fig. 1B), compared with 11.4 μm in the presence of 5% (w/v) glycerol (2), indicating that glycerol does not explain the differences in the K_d values. They could simply be due to the difference in the ionic strength of the buffers. Additionally Lewis et al. work with a heterologous complex with Alb3 from Pisum sativum and cpSRP43 from Arabidopsis thaliana, whereas we use the homologous complex with both Alb3 and cpSRP43 from Arabidopsis thaliana. Further, Lewis et al. (1) conclude that A3CT interacts with the ankyrin repeats of cpSRP43 whereas we showed that A3CT interacts with the chromodomains 2 and 3 (CD2CD3) (2). To support our data, we performed analytical size exclusion chromatography and observed a shift to smaller elution volume for CD2CD3 in the presence of A3CT, but not for cpSRP43Δ2Δ3 lacking CD2CD3 (Fig. 2). This indicates that A3CT binds to CD2CD3 and not to the ankyrin repeats and supports our conclusions from the ITC experiments (2).

FIGURE 1.

Analyses of the effect of glycerol on the K_d of the cpSRP43-A3CT complex using ITC. A, binding isotherms measured by ITC at 20 °C for the titration of ITC Buffer into ITC Buffer. No significant heat change associated with the injections was observed. ITC Buffer: 20 mm Hepes/NaOH pH 7.5, 200 mm NaCl, 2 mm MgCl₂, 1 mm EDTA, 5% (w/v) glycerol, 0.25 mm tris(2-carboxyethyl)phosphine). B, determination of binding affinities for the cpSRP43-A3CT interaction by ITC in the absence of glycerol. The titration was performed at 20 °C in ITC Buffer without glycerol. Analysis of the titration isotherms resulted in a K_d of 13.2 μm and a stoichiometry of 1:1. Data analysis was performed using the Origin 7.0 software.

FIGURE 2.

The C terminus of Alb3 binds to the chromodomains 2 and 3 of cpSRP43. CD2CD3, A3CT, cpSRP43Δ2Δ3, and mixtures of CD2CD3 and A3CT (molar ratio 1:2) or cpSRP43Δ2Δ3 and A3CT (molar ratio 1:2) were subjected to analytical size exclusion chromatography in 20 mm Hepes/NaOH pH 7.5, 200 mm NaCl, 2 mm MgCl₂, 1 mm EDTA, 1 mm DTT using a Superdex200 (10/300) column (shown on the left). Protein-containing fractions were analyzed by SDS-PAGE and are shown on the right. In the presence of A3CT, CD2CD3 shifts to smaller elution volume indicating complex formation between A3CT and CD2CD3.