Possible involvement of a villin-like protein in chloroplast anchoring in spinach mesophyll cells

Chloroplasts maintain specific intracellular distribution patterns in response to environmental conditions to maximize photosynthetic performance and prevent photodamage. We previously proposed that a chloroplast anchoring mechanism functions to fix chloroplasts at appropriate positions and reported that this machinery is actin-dependent and regulated by Ca²⁺ (Takamatsu and Takagi 2011). In the present study, we examined the possible involvement of villin, a Ca²⁺-sensitive actin-binding protein (ABP), in chloroplast anchoring in spinach mesophyll cells. Two antibodies against lily villin proteins, anti-P135ABP and anti-P115ABP, recognized polypeptides of 135 and 120 kDa, respectively, in spinach leaves. These proteins were purified as Ca²⁺-sensitive ABPs by DNase I affinity chromatography. Immunolocalization analysis revealed distinct labeling patterns for the two proteins. Whereas immunostaining with the anti-P115ABP antibody produced randomly distributed punctate signals in the cytoplasm, the 135-kDa villin-like protein was preferentially localized around chloroplasts and was also detected on isolated chloroplasts. Biochemical analyses further revealed that both villin-like proteins bound phosphatidic acid (PA) and phosphoinositides. Functional analysis further showed that addition of the anti-P135ABP antibody induced detachment of a substantial number of chloroplasts and reduced actin filaments on plasma membrane (PM) ghosts. These results indicate that at least two distinct Ca²⁺-sensitive villin-like proteins exist in spinach leaves and suggest that the 135-kDa isoform contributes to chloroplast anchoring by maintaining actin organization around chloroplasts.