Yang X F, Crine P, Boileau G
Département de biochimie, Faculté de médecine, Université de Montréal, Quebec, Canada.
Biochem J. 1995 Nov 15;312 ( Pt 1)(Pt 1):99-105. doi: 10.1042/bj3120099.
Type II integral membrane proteins are anchored by a signal-peptide/membrane-anchor domain (SA domain) located near their N-terminus, whereas type I membrane proteins are anchored by stop-transfer sequences usually located near the C-terminus. In this study we have attempted to transform neutral endopeptidase-24.11 (EC 3.4.24.11; NEP), a type II membrane protein, into a type I membrane protein. Three type I mutant proteins were constructed by fusion of topogenic sequences to the C-terminus of SecNEP, a soluble form of NEP. The first two type I mutants, SecNEP-TMC and SecNEP-TMIC, were constructed by fusing in frame the cytosolic and SA domains of NEP to the C-terminus of SecNEP. These two fusion proteins differ only in the orientation of the cytosolic tail. The third type I mutant, SecNEP-ACE, was constructed by fusing in frame the stop-transfer and cytosolic domains of angiotensin I-converting enzyme (EC 3.4.15.1; ACE) to the C-terminus of SecNEP. Our results suggest that: (1) the NEP ectodomain can be anchored with a type I topology in the endoplasmic reticulum (ER) membrane by both NEP and ACE topogenic sequences; (2) SecNEP-TMC and SecNEP-TMIC were transport-incompetent and needed proteolytic cleavage in the C-terminal region to leave the ER, whereas SecNEP-ACE was transported out of the ER as a type I membrane protein. Therefore we concluded that the nature of topogenic sequences determines the transport-competence of topological mutants of neutral endopeptidase-24.11.
II型整合膜蛋白通过位于其N端附近的信号肽/膜锚定结构域（SA结构域）进行锚定，而I型膜蛋白则通过通常位于C端附近的停止转移序列进行锚定。在本研究中，我们试图将II型膜蛋白中性内肽酶-24.11（EC 3.4.24.11；NEP）转化为I型膜蛋白。通过将拓扑形成序列融合到NEP的可溶性形式SecNEP的C端，构建了三种I型突变蛋白。前两种I型突变体SecNEP-TMC和SecNEP-TMIC是通过将NEP的胞质结构域和SA结构域读框融合到SecNEP的C端构建的。这两种融合蛋白仅在胞质尾的方向上有所不同。第三种I型突变体SecNEP-ACE是通过将血管紧张素I转换酶（EC 3.4.15.1；ACE）的停止转移结构域和胞质结构域读框融合到SecNEP的C端构建的。我们的结果表明：（1）NEP胞外结构域可通过NEP和ACE拓扑形成序列在内质网（ER）膜中以I型拓扑结构进行锚定；（2）SecNEP-TMC和SecNEP-TMIC运输无能力，需要在C端区域进行蛋白水解切割才能离开ER，而SecNEP-ACE作为I型膜蛋白被转运出ER。因此，我们得出结论，拓扑形成序列的性质决定了中性内肽酶-24.11拓扑突变体的运输能力。
