Identification of two regions in the N-terminal domain of ActA involved in 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 elongation 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 present the first evidence that this domain contains all the necessary elements for actin tail formation. A detailed analysis of ActA variants, in which small fragments of the N-terminal region were deleted, allowed the identification of two critical regions. Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process. The first region (region 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 when motility assays are performed in the presence of anti-region T antibodies. The second region (region C, amino acids 21-97), is more specifically involved in maintenance of the continuity of the process, probably by F-actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21-97) leading to 'discontinuous' actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33-74 can interact with F-actin. Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N-terminal domain of ActA.
Lasa
I
I
Unité des Interactions Bactéries-Cellules, Institut Pasteur, Paris, France.
Gouin
E
E
Goethals
M
M
Vancompernolle
K
K
David
V
V
Vandekerckhove
J
J
Cossart
P
P
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002465:Cell Movement; D017124:Conserved Sequence; D017931:DNA Primers; D005260:Female; D008089:Listeria monocytogenes / Q000502:physiology*; D008264:Macrophages; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010063:Ovum / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014982:Xenopus laevis; D001616:beta-Galactosidase / Q000378:metabolism
D000199:Actins; D001426:Bacterial Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C072980:actA protein, Listeria monocytogenes; D001616:beta-Galactosidase