Failure of E. coli K-12 to transport PhoE-LacZ hybrid proteins out of the cytoplasm.

Abstract

A phoE‐lacZ hybrid gene encoding the N‐terminal 300 amino acid residues of pre‐PhoE protein, fused to an almost complete beta‐galactosidase molecule was constructed in vitro. Cell fractionation experiments suggested that the hybrid gene product is transported to the outer membrane. However, by using immuno‐cytochemical labelling on ultra‐thin cryosections it was shown that the hybrid protein accumulated in the cytoplasm. Thus, it appears that: (i) data on the localization of hybrid proteins merely based on cell fractionation experiments are not reliable, and (ii) either the C‐terminal 15% of PhoE protein contain information which is essential for transport, or PhoE‐LacZ hybrid proteins can never be transported out of the cytoplasm. The implications of these results for current models on the translocation of outer membrane proteins are discussed.