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 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 HA-tagged Cps2C protein in D39. Incubation of both protoplasts and membranes with the CP-B resulted in complete degradation of HA-Cps2C in all cases, indicating that the C-terminus of Cps2C was likely extra-cytoplasmic, and hence the protein's topology was not as predicted. Similar results were seen with membranes from TIGR4, indicating 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 extra-cytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this affect 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.