Response to Falk and Sinning: The C Terminus of Alb3 Interacts with the Chromodomains 2 and 3 of cpSRP43

Abstract

This is a response to a letter by Falk and Sinning (1) We recently identified the ankyrin region of cpSRP43 as the primary domain responsible for binding Alb3-Cterm during light-harvesting chlorophyll-binding protein (LHCP) targeting, an interaction shown to facilitate cpSRP43-dependent stimulation of cpSRP GTPases by Alb3-Cterm (2). Falk et al. (3), using only protein interaction assays, report that CD2CD3 of cpSRP43 forms the Alb3-Cterm binding interface, which appears inconsistent with the fact that CD3 is not required for LHCP integration (4) and that CD2 is not required for LHCP integration by a cpSRP54/cpFtsY-independent pathway that relies on cpSRP43/Alb3 (5). We suggested that buffer choice, including the use of glycerol, may play a role in why Falk et al. (3) observed μm rather than nm affinity for cpSRP43 constructs (2). The use of high concentrations of glycerol in isothermal titration calorimetry (ITC) is known to cause experimental artifacts (6). Our control experiments clearly show that the use of glycerol, even at 5% v/v, causes significant background heat changes (Fig. 1). In their letter, Sinning and Falk report a 13 μm affinity even in the absence of glycerol, suggesting glycerol may not be the primary cause for the reported differences. Although species-specific differences in Alb3-Cterm could explain the observed affinity differences, comparing GTPase stimulation by Arabidopsis and Pisum sativum Alb3-Cterm does not support this possibility (Fig. 2). FIGURE 1. Isothermal titration calorimetry investigation of the influence of glycerol in various buffers in buffer to buffer experiments. ITC was conducted by injecting a specific buffer/glycerol formulation into a sample well containing the same buffer/glycerol ... FIGURE 2. Comparison of the ability of Pisum sativum and Arabidopsis thaliana Alb3-Cterm peptide to stimulate cpSRP43-dependent GTP hydrolysis by the cpSRP GTPases. The effect of Alb3-Cterm on the GTP hydrolysis activity of cpSRP54 and cpFtsY was examined in the ... Published reports (2, 4, 5) supporting the physiological relevance of high affinity protein interactions still suggest that the low affinity of cpSRP43 for Alb3-Cterm reported by Falk et al. (3) stems from assay conditions unfavorable for observing the primary targeting interaction that takes place between Alb3-Cterm and the Ank region of cpSRP43. Importantly, buffers used by Falk et al. (3) in ITC and size-exclusion experiments do not support LHCP integration (Fig. 3). In addition, Ank-containing cpSRP43 constructs, including those that lack CD2 and/or CD3, are able to prevent binding of radiolabeled cpSRP43 to Alb3 in salt-washed thylakoids whereas chromodomains do not (Fig. 4). FIGURE 3. Buffer influence on LHCP integration. Salt-washed thylakoids in IBM were incubated with 5 mm ATP, 1 mm GTP, 12.5 μl of radiolabeled pLHCP translation product, and recombinant cpSRP43, cpSRP54, and cpFtsY (2). The final volume was brought to 150 ... FIGURE 4. Competition for cpSRP43 binding to the C terminus of Alb3 in salt-washed thylakoids. Salt-washed thylakoids (equivalent to 75 μg of chlorophyll) and 8 nmol of recombinant cpSRP43 construct as indicated were incubated for 15 min at 25 °C ...