Identification of plant actin-binding proteins by F-actin affinity chromatography

Proteins that interact with the actin cytoskeleton often modulate the dynam ics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been id entified and most are thought to modulate cytoskeleton function. To identif y actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monom ers (globular actin, G-actin) or actin filaments (filamentous actin, F-acti n) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tis sue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovin e serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when elu ted from the G-actin and F-actin columns, respectively, leading to the iden tification of a putative F-actin-binding protein of approximately 40 kDa. W hen plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross- reacted with antiserum against pea annexins. Additionally, a protein that b inds auxin transport inhibitors, the naphthylphthalamic acid binding protei n, which has been previously suggested to associate with the actin cytoskel eton, was eluted in a single peak from the F-actin column. These experiment s provide a new approach that may help to identify novel actin-binding prot eins from plants.