The membrane domain of a bacteriophage assembly protein. Transmembrane-directed proteolysis of a membrane-spanning fusion protein.
A tripartite fusion construct encoding the amino-terminal half of EcoRI endonuclease followed by amino acids 217-299 of the filamentous bacteriophage gene I protein (pI) attached to the enzymatic portion of alkaline phosphatase results in the production of two proteins. The larger protein, pIf, is the complete tripartite fusion protein while the smaller protein, pIf*, results from internal initiation of translation at pI methionine 241. Both pIf and pIf* span the Escherichia coli inner membrane via a 20-amino-acid hydrophobic stretch of pI with their amino termini in the cytoplasm and their carboxyl-terminal alkaline phosphatase domains in the periplasm. The alkaline phosphatase moiety of approximately 70% of pIf is released into the periplasm by in vivo proteolysis, but only about 10% of pIf* is cleaved. Neither DegP, OmpT, nor protease III are responsible for the cleavage in vivo, and leader peptidase is unable to cleave the fusion protein in vitro. Deletion and substitution analyses demonstrate that the degree of periplasmic cleavage depends on the sequence of the cytoplasmic domain of the fusion proteins. Possible mechanisms for this transmembrane-directed cleavage event are compared to proposed models for signal transduction.
Guy-Caffey
J K
JK
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Webster
R E
RE
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D010957:Plasmids; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015246:Deoxyribonuclease EcoRI; D000469:Alkaline Phosphatase