Actin isoforms and post-translational modifications

Isoforms of actin and post-translational modifications. A 2D-PAGE analysis combined with MALDI/TOF detection could verify the expression of different actin isoforms in D. discoideum. The expressed actin isoforms differ in their amino acid sequences, which causes different electrophoretic properties, that resolves the isoforms into distinct spots on a

2D-PAGE. In addition to the genetically encoded differences of actin isoforms, the electrophoretic properties are also influenced by post-translational modifications. These modifications give the spot pattern another layer of complexity, especially as post- translational modification might only affect a subpopulation of any expressed actin isoform. For the actin15 population, the obtained peptide masses might suggest that actin15 is phosphorylated on several, so far, not described residues. These potential phosphorylation sites are residues that are accessible at the surface of the actin molecule. The other possible post-translational modifications were acetylation and arginylation. Unfortunately, the MALDI/ TOF method we used was not suitable to resolve the N-terminal part of the actin molecules, where we would expect these modifications to occur. Very recently, a sensitive sequencing protocol has been described to identify especially N-terminal arginylated peptides using an Orbitrap system (Xu et al., 2009). Certainly, this new approach, combined with the now annotated 2D-gel actinome of D. discoideum, could, in the future, be of great importance to identify N-terminal arginylation in the different actin isoforms. A promising aspect in this line is that the actinome of cells lacking the arginyl-transferase ate1 looked strikingly different on 2D-gels. The main difference we could identify was that the actin comet tail, normally observed in the wild type, is drastically reduced in the ate1 knockout. This is a possible indication that Ate1 in D. discoideum affects actin directly as was suggest for mouse (Wong et al., 2007). An important issue for the future will be to confirm that the lack of the actin comet tail is a direct consequence of the absent arginylation. Of course other possibilities would be that ate1 arginylates upstream effectors of the actin cytoskeleton, that either change the phosphorylation pattern of actin or which influence the expression profiles of the different actin isoforms, that normally constitute the observed actin comet tail.

Localization of GFP-Ate1 in D. discoideum. GFP-Ate1 in D. discoideum localizes mainly in the cytosol. In addition, GFP-Ate1 was also present in the nucleus and flat protrusions, rather untypical for this amoeba. For the mouse it has been reported that the two different Ate1 splice isoforms localize differently (Kwon et al., 1999). The ATE1-2p isoform is exclusively cytosolic, whereas ATE1-1p is present in either the nucleus or the cytosol (Kwon et al., 1999). So the D. discoideum Ate1 ortholog seems more similar to the ATE1- 2p in respect to localization. In addition the localization of GFP-Ate1 to these atypical flat protusions, seems to indicate that in D. discoideum this enzyme catalyzes arginylation in

actin rich structures. The ectopic expression of Ate1 appears to affect the actin cytoskeleton in such a way that flat protusions are formed.

Size of ate1 null cells. The most striking phenotype of ate1 null cells was, that their size is considerably reduced. For mouse it is known that more than 733 cellular proteins are arginylated in vivo (Wong et al., 2007) including cytoskeleton components and components of the house keeping machinery involved in glycolysis, nucleotide biosynthesis, chromatin remodelling/transcription etc. Whether the reduced cell size of the ate1 knockout is due to a direct effect on actin or the result of a more global cause by the lack of arginylation is unclear. It has been reported that a knockout of clathrin, which should severely affect a multitude of vesicle traffic, increases the size of D. discoideum cells. Most likely Ate1 influences a large number of proteins, so that the rather unspecific phenotype of the decreased size, can not be attributed solely to changes in the cytoskeleton.

Contact area of ate1 null. The adhesion areas of ate1 null cells are significantly smaller as judged by RICM. However, preliminary observations suggest that cell-surface adhesion seems not dramatically affected, as knockout cells attach to the surface and show normal cell motility. Additionally, chemotacting ate1 null cells move normal in a cAMP gradient. The cell-surface contact in wild type cells is normally characterized by spotty structures that stain positive for the F-actin probe GFP-LimEΔcoil. In the ate1 knockout the number of these very dynamic F-actin structures seems noticeably reduces. However, ate1 null cells are able to form these actin structures when flattened with an agar slice. We conclude that because of the reduced contact area ate1 null cells show less adhesion points.

Taken together, the initial characterization of an Ate1-like protein in D. discoideum indicates that this enzyme has an effect on the cellular actin. The knockout results in smaller cells with reduced cell-surface areas. Future work should establish if this is a direct consequence of an altered actin arginylation, or rather a response to a cell-wide change in protein arginylation. One stepping stone on this road could be the expression and purification of the recombinant DdAte1 for in vitro arginylations of actin. Assays for in vitro arginylations are already established since Ate1 was first isolated from mammals (Soffer, 1968; Takao et al., 1999). The polymerization kinetics of an arginylated actin population would help to understand the role of this interesting enzyme and the regulation of the actin cytoskeleton.