title: Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.
abstract: A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: J L
authors.initials: JL
authors.lastname: San Millan
authors.forename: D
authors.initials: D
authors.lastname: Boyd
authors.forename: R
authors.initials: R
authors.lastname: Dalbey
authors.forename: W
authors.initials: W
authors.lastname: Wickner
authors.forename: J
authors.initials: J
authors.lastname: Beckwith
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure; D008565:Membrane Proteins / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000648:ultrastructure; D015183:Restriction Mapping; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship
chemical_list: D004251:DNA Transposable Elements; D008565:Membrane Proteins; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase