Subcellular localization of a PhoE-LacZ fusion protein in E. coli by protease accessibility experiments reveals an inner-membrane-spanning form of the protein

Abstract

Protease accessibility experiments were employed to localize a PhoE-LacZ hybrid protein, encompassing a large N-terminal fragment of the outer membrane PhoE protein of E. coli, fused to β-galactosidase, at the subcellular level. In previous studies, this protein was shown to co-fractionate with the outer membrane, whereas immunocytochemical methods suggested a cytoplasmic location. The present results confirm the latter localization. Moreover, it appears that a minor amount of hybrid protein spans the inner membrane, with the PhoE moiety in the periplasm and the β-galactosidase moiety in the cytoplasm. These membrane-spanning proteins might be responsible for the lethal jamming of the export machinery, observed upon induction of synthesis of the protein.