Whittall Jonathan J, Morona Renato, Standish Alistair J
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia.
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia
J Bacteriol. 2015 Jan 1;197(1):120-7. doi: 10.1128/JB.02106-14. Epub 2014 Oct 13.
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.
在革兰氏阳性菌中，酪氨酸激酶被分为两种蛋白质，即细胞质酪氨酸激酶和跨膜衔接蛋白。在肺炎链球菌中，这种跨膜衔接蛋白是CpsC，CpsC的C末端对于CpsD的相互作用及随后的酪氨酸激酶活性至关重要。拓扑结构预测表明，CpsC有两个跨膜结构域，N末端和C末端位于细胞质中。为了研究CpsC的拓扑结构，我们在肺炎链球菌D39菌株中使用了染色体血凝素（HA）标记的Cps2C蛋白。用羧肽酶B（CP-B）处理原生质体和细胞膜，在所有情况下HA-Cps2C均完全降解，这表明Cps2C的C末端可能位于胞外，因此该蛋白的拓扑结构与预测的不同。在肺炎链球菌TIGR4菌株的细胞膜上也观察到了类似结果，表明Cps4C也具有相似的拓扑结构。HA-Cps2C和Cps2D的染色体编码融合蛋白不会被CP-B降解，这表明该融合将C末端固定在了细胞质中。然而，这种融合并未改变荚膜合成。通过流式细胞术检测CpsC的C末端表明，约30%的细胞中其位于胞外。有趣的是，蛋白酪氨酸磷酸酶CpsB的突变体中有显著更多比例的阳性细胞，尽管这种效应与其磷酸酶活性无关。我们的数据表明，CpsC具有多种拓扑结构，其C末端会翻转穿过细胞质膜，在那里它与CpsD相互作用以调节酪氨酸激酶活性。
