Last year we established that binding of Acanthamoeba and Dictyostelium class-I myosins to acidic phospholipid vesicles could be attributed to a short sequence of basic and hydrophobic amino acids, in no specific order, in the basic domain of the tail. We also developed a computer search program to identify similar BH regions, i.e. potenital membrane-binding sites, in other proteins, and demonstrated that BH-search correctly identified all previously known, non-structured membrane-binding sites in 16 proteins. BH-search also identified previously unknown membrane-binding sites in multiple proteins, and synthetic peptides and protein fragments containing these sequences were shown to bind to acidic phospholipid vesicles. Dictyostelium provides an excellent model system for testing the application of these results in living cells. Specifically, we are determining the localization of expressed GFP-labeled wild-type and mutated Dictyostelium myosin IB (DMIB) in DMIB-null cells at different stages of growth and development. Current findings show that the BH-region of the DMIB tail, residues 801KKKVLVHTLIRR812, is both necessary and sufficient for binding DMIB to the plasma membrane of vegetative (growing) amoebae. Deletion of the basic region or mutation or substitution of Ala for the hydrophobic amino acids completely blocks membrane binding. When cells are starved, full-length DMIB dissociates from the plasma membrane, becoming uniformly distributed in the cytoplasm, and then localizes with actin at the front of the motile cell. However, expressed tail alone remains tightly bound to the plasma membrane. Thus, the localization of DMIB is dynamically regulated: the BH region in the basic domain of the tail is required and sufficient for binding DMIB to the plasma membrane of non-polarized, vegetative cells, but the motor domain is required for release of DMIB from the plasma membrane and re-localization within the cytoplasm of motile and chemotaxing cells.
