CHARACTERIZATION OF MAIZE (ZEA-MAYS) POLLEN PROFILIN FUNCTION IN-VITRO AND IN LIVE CELLS

Profilin is a small, 12-15 kDa, actin-binding protein that interacts w ith at least three different ligands. The 1:1 interaction of profilin with globular actin (G-actin) was originally thought to provide a mech anism for sequestering actin monomers in the cytoplasm. It has recentl y become clear that the role of profilin in the cell is more complex, perhaps due to interactions with polyphosphoinositides and proline-ric h proteins, or due to the ability to lower the critical concentration for actin assembly at the fast-growing barbed end of actin filaments. Because actin-binding proteins have been shown to behave differently w ith heterologous sources of actin, we characterized the interaction be tween maize pollen profilins and plant G-actin. The equilibrium dissoc iation constants measured by tryptophan fluorescence quenching were si milar to those of other CaATP-G-actin-profilin complexes (K-d=1.0-1.5 mu M). The ability of maize profilin soforms to bind poly-L-proline wa s analysed, and the K-d values for recombinant pollen and human profil ins were similar when determined by two independent methods. However, the affinity of native maize pollen profilin for poly-L-proline was su bstantially lower than that of any of the recombinant proteins by one of these assays. The possibility of post-translational modification of profilin in the mature pollen grain is discussed. Finally, we quantif ied the effects of microinjection of each profilin isoform on the cyto architecture of Tradescantia stamen hair cells and show that the resul tant disruption can be used to compare actin-binding proteins in livin g cells. The results are discussed in relation to a recent model of th e interphase actin array in these plant cells.