Identification of a Putative Binding Site for a Sea Urchin 53‐kD Myosin Binding Proteina

Abstract

The actomyosin cytoskeleton provides the structural framework for an array of directed cellular movements such as cytokinesis and morphogenesis. Myosin is possibly the major mechanochemical transducer in such movements. The exact structural state of myosin in nonmuscle cells is unknown and may involve cycles of assembly and disassembly.’ Therefore, the regulation of myosin structure and distribution in cells may have a fundamental physiologic role. In a number of cells the redistribution of myosin has been observed to correlate with changes in cell state.’,’ Myosin’s catalytic activity resides in the head of the molecule, and phosphorylation of myosin light chains in this region mediates conformational changes necessary for the regulation of activity. The formation of bipolar filaments is a function of the rod portions of myosin heavy chain. The head-rod junction of myosin heavy chain contains the binding sites for the light chains and plays a role in conformational changes;’ This head-rod junctional region retains a large degree of flexibility. Thus, the neck region determines myosin’s shape and, consequently, filament assembly and enzymatic activity?’ We have identified a myosin-binding protein in sea urchin eggs that mediates myosin’s low ionic strength solubility. This protein has the subunit molecular weight of 53 kD and binds to myosin in a nucleotide-dependent manner. In this study the 53K binding site on myosin was examined to determine the mechanism of interaction that gives rise to a change in myosin’s properties. We used several methods to identify the 53K binding site on the myosin molecule (summarized in FIGURE 1). Myosin and 53K were isolated from soluble sea urchin extracts as described by Yabkowitz and Burgess6 Papain and chymotrypsin digests were performed on myosin and the insoluble and soluble fragments were separated according to Barylko et al.’ Papain gives rise to 165, 130, 107, 80, 62 and 24-kD fragments. The 80, 62, and 24kD fragments are head fragments as defined by actin binding and ATPase activity