Dictyostelium discoideumcontains two profilin isoforms that differ in structure and fuction

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Abstract
Two profilin isoforms (profilins I and II) have been purified from Dictyostelium discoideum, using affinity chromatography on a poly(L-proline) matrix; the isoforms could be separated by cation-exchange chromatography on a FPLC system. The gene coding for profilin I was cloned from a Agtll cDNA library using a profilin I-specific monoclonal antibody. The profilin II cDNA was isolated by probing the cDNA library with an oligonucleotide deduced from the N-terminal amino acid sequence of profilin II, which has an open N terminus in contrast to profilin I. The deduced amino acid sequences of both genes show that profilin I in comparison to profilin II is slightly larger (13 064 Da vs 12 729 Da), has a more acidic isoelectric point (calc, pl 6.62 vs 7.26) and shares with profilin II 68 identical residues out of 126 amino acids. Although both profilins contain a conserved lysine residue in the putative actin-binding region and can be crosslinked covalently to G-actin, the crosslinking efficiency of profilin II to actin is substantially higher than that of profilin I. These data are in agreement with studies on the functional properties of the profilin isoforms. In most prep arations profilin II was more efficient in delaying the onset of elongation during the course of actin polymerization and caused a higher critical concentration for actin polymerization than profilin I, probably due to the slightly increased affinity of profilin II for D. discoideum G-actin (approx. Kd l.8×10-6M) as compared to that of profilin I (approx. Kd 5.l× 10-6M). This difference was not always so obvious and, we are trying to identify non-protein-aceous co-purifying inhibitors that can obscure the data obtained from in vitro assays. The messenger RNA of both profilin isoforms is present throughout development of D. discoideum. However, whereas the profilin I-specific mRNA accumulates after 6-9 h of starvation (aggregation stage), the profilin II-specific mRNA peaks at early stages and disappears during later development almost completely. This suggests differential functions for the profilin isoforms during D. discoideum development.