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 with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose. Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal 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 for further in vivo studies.
Wittenmayer
N
N
Cell Biology Group, Zoological Institute, Technical University, Braunschweig, Germany.
Rothkegel
M
M
Jockusch
B M
BM
Schlüter
K
K
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D003285:Contractile Proteins / Q000378:metabolism; D004926:Escherichia coli; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D051304:Profilins; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism*
D000199:Actins; D003285:Contractile Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; C495739:Pfn1 protein, mouse; D051304:Profilins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins