We have discovered the first protein to bind to a nonfilamentous myosin, aside from actin. This protein, Acan125, is a 125-kDa protein from Acanthamoeba that associates with the SH3 domain of Acanthamoeba myosin-IC and not the SH3 domain of human fodrin. Antibodies raised against Acan125 recognize a single protein of 125 kDa from a whole cell lysate of Acanthamoeba; antibodies to myosin-I (M1.7 and M1.8) do not recognize Acan125 on the same blot. Double labeling of Acanthamoeba show Acan125 and myosin-I to be present on the same intracellular organelle, most likely amoebastomes. Immunoprecipitation with either anti-myosin-I or anti-Acan125 antibodies coprecipitates both Acan125 and myosin-I from a lysate of Acanthamoeba, demonstrating that Acan125 interacts with native myosin-I.Binding through the Src homology domain, SH3, 1 is a recognized means of linking signal transduction proteins (1-3), but a function has not been ascribed to the SH3 domain of the cytoskeletal protein myosin-I. The isoforms of myosin-I that contain an SH3 domain include myosin-Is from Acanthamoeba (4), Dictyostelium (5), Saccharomyces (6), rat (7), and human (8). In the known myosin-I sequences, the SH3 domain invariably resides in tandem with one or two proline-rich domains (5, 9); a proline-rich sequence of 3BP1 has been identified as a motif that binds to the SH3 domain of Abl (10). An interaction has not been demonstrated between SH3 and proline-rich domains of myosin-I, but proline-rich domains of myosin-I have been shown to interact with actin (11-13). These results suggested to us that the SH3 domain of myosin-I might be available for interaction with another protein.The proposal of proteins that interact with myosin-I is rooted in efforts to reconcile reconstitution results with cellular localization studies. In vitro binding (11,14,15) and motility (16) assays are consistent with myosin-I acting mechanically on the surface of any membrane containing the ubiquitous phospholipid phosphatidylserine. But immunostaining shows myosin-I to be excluded from most cell membranes and to be concentrated at the leading edges of migrating cells (17, 18) and on selected organelles (19). The contractile vacuole of Acanthamoeba has been demonstrated to selectively bind the myosin-IC isoform (20). Myosin-IA and myosin-IB were found, using immunogold, to be associated along one side of fractionated membranes (21), as though bound to proteins.Myosin-I could associate with other proteins on membrane surfaces via interactions with SH3. SH3 domains have been shown to mediate specific associations between SH3-containing proteins and various binding partners, including phosphatidylinositol 3-kinase (22-25), p22 phox , and p47phox (26 -28), and dynamin (29 -31). In each of these studies, bacterially expressed fusion proteins of SH3 domains were used as affinity ligands to selectively extract the binding partner from a cell lysate. Selectivity may be dictated by the structures of both the SH3 domain and its binding partner (32,33). Thus, binding partners fo...