title: Application of a Saccharomyces cerevisiae model to study requirements for trafficking of Yersinia pestis YopM in eucaryotic cells.
abstract: YopM is a leucine-rich repeat (LRR) virulence protein that is delivered into host cells when any of the three human-pathogenic species of Yersinia binds to mammalian cells. It exhibits heterogeneity of size and sequence among the yersiniae, but the functional consequences of this variability are not yet known. Yersinia pestis YopM was previously shown to accumulate in the nuclei of infected HeLa cells by a mechanism that requires vesicular trafficking. In this study, we characterized the trafficking of Y. pestis YopM in a Saccharomyces cerevisiae model previously found to support nuclear localization of YopM from an enteropathogenic Yersinia strain (C. F. Lesser and S. I. Miller, EMBO J. 20:1840-1849, 2001). Y. pestis YopM was N-terminally fused to the yeast enhanced green fluorescent protein (yEGFP) and inducibly expressed in the cytoplasm. yEGFP-YopM localized to the yeast nucleus, showing that this property is conserved for YopMs so far tested and that infection and the presence of other Yops are not required for its trafficking. When expressed in S. cerevisiae that is temperature sensitive for vesicular transport, YopM failed to accumulate in the nucleus at the nonpermissive temperature but did accumulate when the permissive temperature was restored. This shows that vesicular trafficking also is required in yeast for normal localization of YopM. YopM consists of a 71-residue leader sequence, 15 LRRs, and a 32-residue tail. Deletion analysis revealed that the leader sequence or tail is alone insufficient to direct YopM to the nucleus, showing that the LRR structure is required. Both the N-terminal and C-terminal halves of YopM localized to the nucleus, indicating the possible presence of two nuclear localization signals (NLSs) in YopM or domains in YopM where an NLS-containing protein might bind; this fits with the presence of two highly conserved regions among Yersinia YopMs. yEGFP-YopM lacking LRRs 4 to 7 or 7 to 10 accumulated in the nucleus in yeast, and YopM lacking these LRRs concentrated normally in the HeLa cell nucleus after delivery by Yersinia infection, showing that these LRRs are not essential for YopM trafficking in eucaryotic cells. However, because Y. pestis carrying either of these YopMs is strongly compromised in virulence in mice, these findings revealed that LRRs 4 to 10 map a region of YopM or support a conformation of YopM that is necessary for a pathogenic effect.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington 40536-0298, USA.
authors.forename: Elbieta
authors.initials: E
authors.lastname: Skrzypek
authors.forename: Tanya
authors.initials: T
authors.lastname: Myers-Morales
authors.forename: Sidney W
authors.initials: SW
authors.lastname: Whiteheart
authors.forename: Susan C
authors.initials: SC
authors.lastname: Straley
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015010:Yersinia pestis / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; C060772:yopM protein, Yersinia; D049452:Green Fluorescent Proteins