Vinculin is an adhesion plaque component localized on the cytoplasmic side of the cell membrane where stress fibers end. To detect vinculin-binding proteins, we have developed an 1251-vinculin gel overlay method. SDS PAGE was used to separate different protein preparations. After fixing the proteins in the gel with methanol-acetic acid, the SDS was removed with ethanol and the proteins renatured in buffer. The gel was then incubated with 1251-vinculin. After extensive washing to remove nonspecifically associated label, the gel was dried and autoradiographed. Chick embryo fibroblasts, their Rous sarcoma virus transformants, and HeLa cells were found to contain two proteins (Mr 220,000 and 130,000) that bound 12Sl-vinculin strongly and another (Mr 42,000) that bound it moderately. The 130,000-mol-wt protein was identified as vinculin itself, which suggests that it may self-associate. The 42,000-mol-wt protein was identified as actin with which vinculin is known to interact. The identity of the 220,000-mol-wt protein is not known. It is not cellular fibronectin, myosin, or filamin. When fibroblast proteins were separated into Triton X-100 soluble and insoluble fractions, most of the vinculin and the 220,000-mol-wt protein was found to be in the soluble fraction. Chicken gizzard also contained these vinculin-binding proteins along with three others of Mr 190,000, 170,000, and 100,000.Cells in culture adhere tightly to the substrate at discrete sites called adhesion plaques and less tightly in regions of close contact (1,22). The ends of stress fibers, bundles of actin filaments and associated proteins, are localized on the cytoplasmic side of the adhesion plaque (2). This suggests that adhesive molecules on the cell surface and elements of the actin-based cytoskeleton are physically linked.Recently, Geiger (14) and Burridge and Feramisco (7) identified vinculin, a protein of Mr 130,000, and localized it on the cytoplasmic side of the adhesion plaque. Vinculin interacts with actin (8,23,30) and has been postulated to link the ends of actin filaments to the membrane (7,(14)(15)(16)30). Since vinculin does not appear to be an integral membrane protein (14), there must be at least one transmembane connection to the substratum.To search for such a transmembrane connection as well as other proteins that interact with vinculin and that may be adhesion plaque components, we have developed an 1251-vinculin gel overlay method. In this paper, we describe this method and demonstrate that two proteins found in chick fibroblasts, chicken gizzard, and HeLa cells strongly bind '25I-vinculin. The identity of one of these proteins of 220,000 mol wt is unknown; the other, of 130,000 mol wt is vinculin itself.
MATERIALS AND METHODS
Cells and Cell Preparations:Normal chick embryo flbroblasts (CEF) ~ were prepared by trypsinizing decapitated 12-d-old chick embryos with their internal organs removed. The cells were cultured in Dulbecco's modified Eagle's medium (DME) supplemented with 10% calf serum, 0.125 ug/ml penicillin, and 0...