title: Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.
abstract: 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.
authors.affiliation: Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA.
authors.forename: S
authors.initials: S
authors.lastname: Hu
authors.forename: S R
authors.initials: SR
authors.lastname: Brady
authors.forename: D R
authors.initials: DR
authors.lastname: Kovar
authors.forename: C J
authors.initials: CJ
authors.lastname: Staiger
authors.forename: G B
authors.initials: GB
authors.lastname: Clark
authors.forename: S J
authors.initials: SJ
authors.lastname: Roux
authors.forename: G K
authors.initials: GK
authors.lastname: Muday
mesh_terms: D000199:Actins / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D003285:Contractile Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D018546:Hypocotyl / Q000737:chemistry; D015151:Immunoblotting; D008840:Microfilament Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D010940:Plant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051304:Profilins; D014675:Vegetables / Q000737:chemistry / Q000378:metabolism*; D003313:Zea mays / Q000378:metabolism
chemical_list: D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D010940:Plant Proteins; D051304:Profilins