The early interaction of the outer membrane protein PhoE with the periplasmic chaperone Skp occurs at the cytoplasmic membrane

Spheroplasts were used to study the early interactions of newly synthesized outer membrane protein PhoE with periplasmic proteins employing a protein cross-linking approach. Newly translocated PhoE protein could be cross-link ed to the periplasmic chaperone Skp at the periplasmic side of the inner me mbrane. To study the timing of this interaction, a PhoE-dihydrofolate reduc tase hybrid protein was constructed that formed translocation intermediates , which had the PhoE moiety present in the periplasm and the dihydrofolate reductase moiety tightly folded in the cytoplasm, The hybrid protein was fo und to cross-link to Skp, indicating that PhoE closely interacts with the c haperone when the protein is still in a transmembrane orientation in the tr anslocase. Removal of N-terminal parts of PhoE protein affected Skp binding in a cumulative manner, consistent with the presence of two Skp-binding si tes in that region. In contrast, deletion of C-terminal parts resulted in v ariable interactions with Skp, suggesting that interaction of Skp with the N-terminal region is influenced by parts of the C terminus of PhoE protein. Both the soluble as well as the membrane-associated Skp protein were found to interact with PhoE, The latter form is proposed to be involved in the i nitial interaction with the N-terminal regions of the outer membrane protei n.