title: Transformation of the signal peptide/membrane anchor domain of a type II transmembrane protein into a cleavable signal peptide.
abstract: Rabbit neutral endopeptidase-24.11 is a type II transmembrane protein with a 27-amino acid residue positively charged NH2-terminal cytoplasmic domain, a 23-amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. In order to study the mechanism of membrane anchoring of neutral endopeptidase-24.11, we created mutants in which the cytoplasmic tail was deleted. Expression of these mutants in COS-1 cells resulted in the secretion of approximately 10-20% of the protein into the culture medium, due possibly to the cleavage of part or all of the signal peptide/membrane anchor domain by the rough endoplasmic reticulum signal peptidase. In a second set of mutants, a hydrophilic sequence (GSQNS) was inserted midway in the signal peptide/membrane anchor domain of neutral endopeptidase-24.11. When this hydrophilic sequence was introduced into the full-length neutral endopeptidase-24.11, approximately 20% of the enzyme activity was recovered in the culture medium. This proportion increased to 93% when the cytosolic tail was deleted. Sequencing of the [3H]tyrosine- or [3H]isoleucine-labeled secreted protein indicated that proteolysis, possibly by signal peptidase, occurred on the COOH-terminal side of the signal peptide/membrane anchor domain. We conclude that the efficient cleavage of the signal peptide/membrane anchor domain and secretion of the protein require both the deletion of the cytosolic domain and the presence of a hydrophilic sequence.
authors.affiliation: Département de Biochimie, Faculté de Médecine, Université de Montréal, Québec, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Roy
authors.forename: C
authors.initials: C
authors.lastname: Chatellard
authors.forename: G
authors.initials: G
authors.lastname: Lemay
authors.forename: P
authors.initials: P
authors.lastname: Crine
authors.forename: G
authors.initials: G
authors.lastname: Boileau
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000737:chemistry / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins; D012995:Solubility; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D015260:Neprilysin