Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

• Published in: The Plant journal : for cell and molecular biology 2000-10, Vol.24 (1), p.127-137
• Main Authors: Hu, S., Brady, S. R., Kovar, D. R., Staiger, C. J., Clark, G. B., Roux, S. J., Muday, G. K.
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Cell Membrane - chemistry; Chromatography, Affinity - methods; Contractile Proteins; Electrophoresis, Polyacrylamide Gel; Hypocotyl - chemistry; Immunoblotting; Life Sciences (General); Microfilament Proteins - isolation & purification; Microfilament Proteins - metabolism; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Profilins; Space life sciences; Vegetables - chemistry; Vegetables - metabolism; Zea mays - metabolism
• ISSN: 0960-7412

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine 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 eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When 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 binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.