The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.
The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.
Department of Microbiology, University of Pennsylvania, School of Medicine, Philadelphia 19104-4318, USA.
G A
GA
Smith
J A
JA
Theriot
D A
DA
Portnoy
D000199:Actins / Q000032:analysis / Q000096:biosynthesis; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000032:analysis*; D002460:Cell Line; D003285:Contractile Proteins / Q000032:analysis*; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D007928:Lethal Dose 50; D008089:Listeria monocytogenes / Q000737:chemistry / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008840:Microfilament Proteins / Q000032:analysis*; D009154:Mutation; D010750:Phosphoproteins / Q000032:analysis*; D011108:Polymers; D051304:Profilins; D011392:Proline; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D001426:Bacterial Proteins; D015815:Cell Adhesion Molecules; D003285:Contractile Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D008840:Microfilament Proteins; C495739:Pfn1 protein, mouse; D010750:Phosphoproteins; D011108:Polymers; D051304:Profilins; D000098662:Vasodilator-Stimulated Phosphoprotein; C072980:actA protein, Listeria monocytogenes; D011392:Proline