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 with at least three different ligands. The 1:1 interaction of profilin with globular actin (G-actin) was originally thought to provide a mechanism for sequestering actin monomers in the cytoplasm. It has recently become clear that the role of profilin in the cell is more complex, perhaps due to interactions with polyphosphoinositides and proline-rich 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 with heterologous sources of actin, we characterized the interaction between maize pollen profilins and plant G-actin. The equilibrium dissociation constants measured by tryptophan fluorescence quenching were similar to those of other CaATP-G-actin-profilin complexes (Kd=1.0-1.5 microM). The ability of maize profilin isoforms to bind poly-l-proline was analysed, and the Kd values for recombinant pollen and human profilins were similar when determined by two independent methods. However, the affinity of native maize pollen profilin for poly-l-proline was substantially 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 quantified the effects of microinjection of each profilin isoform on the cytoarchitecture of Tradescantia stamen hair cells and show that the resultant disruption can be used to compare actin-binding proteins in living cells. The results are discussed in relation to a recent model of the interphase actin array in these plant cells.
Gibbon
B C
BC
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Ren
H
H
Staiger
C J
CJ
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism; D002467:Cell Nucleus / Q000648:ultrastructure; D003285:Contractile Proteins; D003593:Cytoplasm / Q000648:ultrastructure; D006801:Humans; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D008845:Microinjections; D010455:Peptides / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D010944:Plants / Q000648:ultrastructure*; D011058:Pollen; D051304:Profilins; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D003313:Zea mays / Q000737:chemistry*
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D010455:Peptides; D010940:Plant Proteins; D051304:Profilins; D011994:Recombinant Proteins; C011083:polyproline