title: Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway.
abstract: Upon infection of mammalian cells, Listeria monocytogenes lyses the phagosome and enters the cytosol, where it secretes proteins necessary for its intracellular growth cycle. Consequently, bacterial proteins exposed to the cytosol are potential targets for degradation by host cytosolic proteases. One pathway for degradation of host cytosolic proteins, the N-end rule pathway, involves recognition of the N-terminal amino acid and is mediated by the proteasome. However, very few natural N-end rule substrates have been identified. We have examined the L. monocytogenes ActA protein as a potential target for this pathway. ActA is an essential determinant of L. monocytogenes pathogenesis that is required to induce actin-based motility and cell-to-cell spread. We show that the half-life of a secreted form of ActA can be altered in the mammalian cytosol by changing the N-terminal amino acid. Moreover, the introduction of a destabilizing N-terminus into the functional, surface-bound form of ActA results in a small-plaque phenotype in L2 cells, which is partially reversible by an inhibitor of the proteasome. These results indicate that the L. monocytogenes ActA protein is a natural N-end rule substrate, and that optimal function of ActA in mediating cell-to-cell spread is dependent upon its intracellular turnover rate.
authors.affiliation: Department of Microbiology, University of Pennsylvania, School of Medicine, Philadelphia 19104-6076, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Moors
authors.forename: V
authors.initials: V
authors.lastname: Auerbuch
authors.forename: D A
authors.initials: DA
authors.lastname: Portnoy
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015169:Colony Count, Microbial; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006207:Half-Life; D007976:Leupeptins / Q000494:pharmacology; D008089:Listeria monocytogenes / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009154:Mutation; D046988:Proteasome Endopeptidase Complex
chemical_list: D001426:Bacterial Proteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; C063261:acetylleucyl-leucyl-norleucinal; C072980:actA protein, Listeria monocytogenes; D001120:Arginine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex