Premium
Systematic mutational analysis of the amino‐terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin‐based motility
Author(s) -
Lauer Peter,
Theriot Julie A.,
Skoble Justin,
Welch Matthew D.,
Portnoy Daniel A.
Publication year - 2001
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2001.02677.x
Subject(s) - biology , actin , motility , listeria monocytogenes , cytoplasm , amino acid , microbiology and biotechnology , gliding motility , cytoskeleton , actin binding protein , actin cytoskeleton , biochemistry , genetics , cell , bacteria
The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged‐to‐alanine mutations in the NH 2 ‐terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin‐binding sequence of ActA spans 40 amino acids. We also identified a ‘motility rate and cloud‐to‐tail transition’ region in which nine contiguous mutations spanning amino acids 165–260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to ‘skid’ or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin‐based motility of L. monocytogenes .