Interaction of Plant Profilin with Mammalian Actin

Abstract

The mode of interaction of birch and bovine profilins with actin was compared using a number of techniques. Birch profilin was purified from pollen or as a recombinant protein from Escherichia coli, using poly(L-proline) affinity chromatography and a monoclonal antibody for the identification of the isolated product. On two-dimensional gels, the genuine and recombinant proteins were identical in molecular mass and isoelectric point and revealed that birch profilin, in contrast to the basic profilins found in mammals, is an acidic protein, analogous to maize profilins. Bovine profilin was obtained from calf thymus.In viscometric assays, the birch protein was seen to modulate actin filament formation analogous to animal profilin. Birch profilin increased the critical concentration required for muscle and brain actin polymerization in a concentration-dependent manner, supporting the notion of the formation of a heterologous complex between the plant protein and animal actin. The effect was Mg*+-sensitive, as had been described for homologous complexes. The dissociation constants obtained for the planthertebrate and the vertebratehertebrate system were both in the micromolar range. The affinity of birch profilin for muscle actin was slightly lower than that for nonmuscle (brain) actin. A binary complex of birch profilin and skeletal muscle actin could be isolated by gel chromatography.Cross-linking experiments with actin, birch profilin, the G-actin binding peptide thymosin p4 and gelsolin segment 1, the N-terminal fragment of an actin capping protein, showed that profilin competed with thymosin p4, but had no effect on segment 1 binding to actin.These data indicate that the actin-binding domains in plant and animal profilins are functionally highly conserved, although the overall sequence similarity is less than 25%.Profilins are small (14-17 kDa), ubiquitous proteins probably involved in the regulation of actin dynamics (cf.

Actin Purified from Maize Pollen Functions in Living Plant Cells.

“…We have recently established that human profilin has a significantly higher affinity for purified pollen G-actin under low-ionic-strength conditions . This is also consistent with the observation that plant profilins have a lower affinity for vertebrate actin isoforms than do bovine spleen or thymus profilin (Giehl et al, 1994;Perelroizen et al, 1996). Although endogenous profilin may be present in equimolar amounts with actin in pollen extracts (Vidali and Hepler, 1997), the low affinity of the interaction may prevent the isolation of large amounts of profilin-actin complex (see Ruhlandt et al, 1994).…”

“…A common approach for dissecting the interaction of ABPs with actin isto approximate the in vivo conditions by combining purified components in vitro. Most physicochemical studies of plant ABPs have used the most readily available source of eukaryotic actin, the rabbit skeletal muscle a-actin isoform (RSMA), and bacterially expressed plant ABPs (Giehl et al, 1994;Lopez et al, 1996;Perelroizen et al, 1996;Carlier et al, 1997). Although studies that have used heterologous components provide compelling evidence that plant ABPs are indeed functional, many important subtleties may be either lost or misinterpreted.…”

“…Analysis of the physicochemical properties of plant ABPs has been greatly facilitated by the ability to overexpress recombinant proteins in bacteria (e.g., Giehl et al, 1994;Lopez et al, 1996;Perelroizen et al, 1996;Carlier et al, 1997). Unfortunately, this strategy has not been successfully applied to any eukaryotic actin (reviewed in Sheterline et al, 1995).…”

Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1)

“…Although some methods have been developed to get relatively high yields of actin from plant pollens [24,25], it is still very difficult to purify actin (especially actin isoforms) from most of the plants [26]. Expression of proteins in E. coli has greatly facilitated our understanding of physicochemical properties of proteins including plant actin binding protein [27,28]. Unfortunately, there is still no successful example of the expression of active eukaryotic actin in E. coli.…”

Epitope Tag-Antibody Combination Useful for the Detection of Protein Expression in Prokaryotic and Eukaryotic Cells

“…It is recognized by the monoclonal anti-birch profilin antibody 4A6, which has been characterized as an immunoglobulin IgG2a (1). Epitope recognition by 4A6 is restricted to profilins of the birch tree family (4) and has been demonstrated in immunoblotting, immunoprecipitation and immunofluorescence (1,3).…”