41001:

The NPA binding protien and annexin-like proteins from zucchini bind to actin filaments.

Authors:	Muday, Gloria, K.(A)Hu, Shiquan(A)Brady, Shari, R.(A)Kovar, David, R.(B)Staiger, Christopher, J.(B)Clark, Greg, B.(C)Roux, Stanley, J.(C)
Affiliations:	(A): Wake Forest University (B): Purdue University (C): University of Texas
Presenter:	Muday, Gloria K., muday@wfu.edu

The actin cytoskeleton is a filamentous network containing cross-linked polymers of globular actin (G-actin) monomers and a myriad of associated proteins. The polymerization and organization of filamentous actin (F-actin) can be dramatically altered to allow changes in growth and development. The actin cytoskeleton is required for localization of membrane proteins, cell motility, cell division, surface remodeling, control of cell shape, development of polarity, movement of vesicles, secretion, and endocytosis. To understand the way that these processes are mediated by the actin cytoskeleton it is necessary to identify the specific actin binding proteins that modulate the cytoskeleton. One powerful approach to identify actin-binding proteins is to create affinity columns with immobilized G- or F-actin. One membrane protein complex that may use the actin cytoskeleton for its polar distribution is the auxin efflux carrier. In stems, this protein complex is localized to the basal plasma membrane and thereby controls the polarity of auxin movement. The auxin efflux carrier contains a regulatory subunit that binds inhibitors that block auxin transport, such as naphthylphthalamic acid (NPA). Previous experiments have demonstrated that the NPA binding protein remains with the cytoskeletal during detergent extraction and partitions with actin during in vitro depolymerization and repolymerization experiments. The purpose of this study was to more directly examine the interaction between the NPA binding protein and actin filaments using F-actin affinity chromatography. Zucchini hypocotyl G-actin was purified to electrophoretic homogeneity and was shown to be native and competent for polymerization to actin filaments. Purified plasma membranes were treated to release actin associated proteins, and the resulting extracts were applied to G-actin, F-actin and BSA affinity columns. Two abundant polypeptides selectively eluted from the F-actin column and cross-reacted with antiserum against pea annexins. NPA binding activity was specifically eluted in a single peak from the F-actin column, but was not retained by a BSA column. Drugs that alter the state of actin polymerization also decreased the amount of polar auxin transport. The function of the association of the NPA binding protein with actin filaments may be to localize the auxin efflux carrier complex in the basal plasma membrane and thereby control the polarity of auxin transport.