Translocation of neutral endopeptidase 24.11 mutants with deletions of the NH2-terminal cytosolic domain.
Rabbit neutral endopeptidase 24.11 (NEP) is a type II membrane protein with a positively charged 27 amino acid residue NH2-terminal cytoplasmic domain, a 20 amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. To study the role of the cytosolic domain in anchoring NEP in the plasma membrane, we constructed two mutants in which this cytosolic domain was deleted. In the first mutant (NEP delta cyto), a Glu residue was present in NH2-terminus, while a Lys residue was substituted at the same position in the second mutant (NEP delta cyto(K)). To better understand the interaction of these mutants with the rough endoplasmic reticulum membrane, the mutated NEP cDNAs were transcribed and translated in vitro in the presence of microsomal membranes. Our studies showed that deletion of the hydrophillic cytosolic domain affects translocation of the NEP polypeptide chain. Substitution of a positively charged Lys residue for the Glu residue at the NH2-terminus of the deletion mutant only partly restored translocation of the polypeptide chain. Furthermore, carbonate extraction and trypsin digestion of the microsomal membranes indicated that the deletion mutants are inserted in the microsomal membranes as type III membrane proteins with their COOH-terminal domain exposed on the exterior of the microsomes. Thus, efficient translocation is dependent on the presence of a charged cytoplasmic domain.
Lemire
I
I
Département de biochimie, Faculté de médecine, Université de Montréal, PQ, Canada.
Roy
P
P
Boileau
G
G
D000595:Amino Acid Sequence; D001692:Biological Transport; D003600:Cytosol / Q000201:enzymology*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D017353:Gene Deletion; D006031:Glycosylation; D007425:Intracellular Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D014176:Protein Biosynthesis
D015260:Neprilysin