title: Topology of Streptococcus pneumoniae CpsC, a polysaccharide copolymerase and bacterial protein tyrosine kinase adaptor protein.
abstract: In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. In Streptococcus pneumoniae, this transmembrane adaptor is CpsC, with the C terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest that CpsC has two transmembrane domains, with the N and C termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal hemagglutinin (HA)-tagged Cps2C protein in S. pneumoniae strain D39. Incubation of both protoplasts and membranes with carboxypeptidase B (CP-B) resulted in complete degradation of HA-Cps2C in all cases, indicating that the C terminus of Cps2C was likely extracytoplasmic and hence that the protein's topology was not as predicted. Similar results were seen with membranes from S. pneumoniae strain TIGR4, indicating that Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C terminus by flow cytometry indicated that it was extracytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this effect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C terminus flipping across the cytoplasmic membrane, where it interacts with CpsD in order to regulate tyrosine kinase activity.
authors.affiliation: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia.
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Whittall
authors.affiliation: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia.
authors.forename: Renato
authors.initials: R
authors.lastname: Morona
authors.affiliation: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia alistair.standish@adelaide.edu.au.
authors.forename: Alistair J
authors.initials: AJ
authors.lastname: Standish
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D011487:Protein Conformation; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D037521:Virulence Factors