Abstract
Intracellular chloroplast positioning is important to optimize photosynthesis. The actin cytoskeleton has been suggested to participate in the chloroplast positioning, however, the manner of participation has not yet been elucidated. To verify our hypothesis that chloroplasts are anchored to the cortical cytoplasm by actin cytoskeleton, we prepared plasma-membrane (PM) ghosts, on which the cytoplasmic side of PM was exposed, from spinach mesophyll protoplasts. Our studies suggested that the attachment of chloroplasts to PM ghosts is actin dependent and regulated by Ca2+-calmodulin. From these results, we assumed participation of villin, an actin-binding protein exhibiting the Ca2+- calmodulin dependent actin-depolymerizing activity. We detected two kinds of villin-like polypeptides in spinach. Both polypeptides were localized in the vicinity of chloroplasts on PM ghosts. In addition, the anti-villin antibodies induced a disorder of actin filaments and detachment of chloroplasts from the PM ghosts.
