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

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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.