Functional characterization of green fluorescent protein-profilin fusion proteins

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared wi th 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to e ither the C-terminus or N-terminus of GFP. These fusion proteins were expre ssed in Escherichia coil and affinity-purified on polyproline-Sepharose. In teraction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P-2. The affinity for actin was quantitatively determined in polymerization assa ys. Our results show that fusion of GFP to the C-terminus of profilin I abo lishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P-2 was not signific antly altered. Furthermore, fusion of either isoform with GFP did not decre ase the affinity for actin. In localization studies with mammalian cells, a ll fusion proteins showed the localization expected for profilin in areas o f high actin dynamics, such as leading lamellae and ruffles induced by epid ermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites. Therefore, other constructs should be preferred fo r further in vivo studies.