The characterization of ligand-specific maize (Zea mays) profilin mutants

Profilins are low-molecular-mass (12 15 kDa) cytosolic proteins that are ma jor regulators of actin assembly in all eukaryotic cells. In general, profi lins from evolutionarily diverse organisms share the ability to bind to G-a ctin, poly-(L-proline) (PLP) and proline-rich proteins, and polyphosphoinos itides. However, the functional importance of each of these interactions re mains unclear and might differ between organisms. We investigated the impor tance of profilin's interaction with its various ligands in plant cells by characterizing four maize (Zea mays) profilin 5 (ZmPRO5) mutants that had s ingle amino acid substitutions in the presumed sites of ligand interaction. Comparisons in vitro with wild-type ZmPRO5 showed that these mutations alt ered ligand association specifically. ZmPRO5-Y6F had a 3-fold increased aff inity for PLP, ZtnPRO5-Y6Q had a 5-fold decreased affinity for PLP, ZmPRO5- D8A had a 2-fold increased affinity for PtdIns(4,5)P-2 and ZmPRO5-K86A had a 35-fold decreased affinity for G-actin. When the profilins were microinje cted into Tradescantia stamen hair cells, ZmPRO5-Y6F increased the rate of nuclear displacement in stamen hairs, whereas ZmPR05K86A decreased the rate . Mutants with a decreased affinity for PLP (ZmPRO5-Y6Q) or an enhanced aff inity for PtdIns(4,5)P-2 (ZmPRO5-D8A) were not significantly different from wild-type ZmPR05 in affecting nuclear position. These results indicate tha t plant profilin's association with G-actin is extremely important and furt her substantiate the simple model that profilin acts primarily as a G-actin -sequestering protein in plant cells. Furthermore, interaction with proline -rich binding partners might also contribute to regulating profilin's effec t on actin assembly in plant cells.