Phage Shock Protein C (PspC) of Yersinia enterocolitica Is a Polytopic Membrane Protein with Implications for Regulation of the Psp Stress Response

Phage shock proteins B (PspB) and C (PspC) are integral cytoplasmic membrane proteins involved in inducing the Yersinia enterocolitica Psp stress response. A fundamental aspect of these proteins that has not been studied in depth is their membrane topologies. Various in silico analyses universally predict that PspB is a bitopic membrane protein with the C terminus inside. However, similar analyses yield conflicting predictions for PspC: a bitopic membrane protein with the C terminus inside, a bitopic membrane protein with the C terminus outside, or a polytopic protein with both termini inside. Previous studies of Escherichia coli PspB-LacZ and PspC-PhoA fusion proteins supported bitopic topologies, with the PspB C terminus inside and the PspC C terminus outside. Here we have used a series of independent approaches to determine the membrane topologies of PspB and PspC in Y. enterocolitica. Our data support the predicted arrangement of PspB, with its C terminus in the cytoplasm. In contrast, data from multiple independent approaches revealed that both termini of PspC are located in the cytoplasm. Additional experiments suggested that the C terminus of PspC might be the recognition site for the FtsH protease and an interaction interface with PspA, both of which would be compatible with its newly proposed cytoplasmic location. This unexpected arrangement of PspC allows a new model for events underlying activation of the Psp response, which is an excellent fit with observations from various previous studies.