Lemire I, Roy P, Boileau G
Département de biochimie, Faculté de médecine, Université de Montréal, PQ, Canada.
Biochem Cell Biol. 1994 May-Jun;72(5-6):182-7. doi: 10.1139/o94-027.
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.
兔中性内肽酶24.11（NEP）是一种II型膜蛋白，具有带正电荷的27个氨基酸残基的NH2末端胞质结构域、20个氨基酸残基的疏水信号肽/膜锚定结构域，以及一个暴露于膜外质侧的大催化COOH末端结构域。为了研究胞质结构域在将NEP锚定在质膜中的作用，我们构建了两个缺失该胞质结构域的突变体。在第一个突变体（NEP delta cyto）中，NH2末端存在一个Glu残基，而在第二个突变体（NEP delta cyto(K)）的相同位置上一个Lys残基被取代。为了更好地理解这些突变体与糙面内质网膜的相互作用，在微粒体膜存在的情况下，对突变的NEP cDNA进行体外转录和翻译。我们的研究表明，亲水胞质结构域的缺失会影响NEP多肽链的转运。在缺失突变体的NH2末端用带正电荷的Lys残基取代Glu残基只能部分恢复多肽链的转运。此外，对微粒体膜进行碳酸盐提取和胰蛋白酶消化表明，缺失突变体作为III型膜蛋白插入微粒体膜中，其COOH末端结构域暴露于微粒体外部。因此，有效的转运依赖于带电荷的胞质结构域的存在。
