IDENTIFICATION OF 2 REGIONS IN THE N-TERMINAL DOMAIN OF ACTA INVOLVEDIN THE ACTIN COMET TAIL FORMATION BY LISTERIA-MONOCYTOGENES

The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface. The continuous process of actin filament elong ation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts. Here, by fusing the N-terminus of ActA (residues 1-234) to the omega fragment of beta-galactosidase, we prese nt the first evidence that this domain contains all the necessary elem ents for actin tail formation. A detailed analysis of ActA variants, i n which small fragments of the N-terminal region were deleted, allowed the identification of two critical regions. Both are required to init iate the actin polymerization process, but each has in addition a spec ific role to maintain the dynamics of the process. The first region (r egion T, amino acids 117-121) is critical for filament elongation, as shown by the absence of actin tail in a 117-121 deletion mutant or whe n motility assays are performed in the presence of anti-region T antib odies. The second region (region C, amino acids 21-97), is more specif ically involved in maintenance of the continuity of the process, proba bly by F-actin binding or prevention of barbed end capping, as strongl y suggested by both a deletion (21-97) leading to 'discontinuous' acti n tail formation and in vitro experiments showing that a synthetic pep tide covering residues 33-74 can interact with F-actin. Our results pr ovide the first insights in the molecular dissection of the actin poly merization process induced by the N-terminal domain of ActA.