Lemire I, Lazure C, Crine P, Boileau G
Départment de biochimie, Faculté de médecine, Université de Montréal, Québec, Canada.
Biochem J. 1997 Feb 15;322 ( Pt 1)(Pt 1):335-42. doi: 10.1042/bj3220335.
Signal peptide/membrane anchor (SA) domains of type II membrane proteins initiate the translocation of downstream polypeptides across the endoplasmic reticulum (ER) membrane. In contrast with signal peptides, however, SA domains are not cleaved by signal peptidase and thus anchor the protein in the membrane. In the present study we have introduced mutations in the SA domain of neprilysin (neutral endopeptidase-24.11; NEP) to identify structural elements that would favour the processing of SA domains by signal peptidase. Mutants of full-length and truncated (without cytoplasmic domain) protein were constructed by substitution of the sequences SQNS, QQTT or YPGY for VTMI starting at position 15 of the NEP SA domain. In addition, a Pro residue was substituted for Thr at position 16 of the SA domain. The rationale for the use of these sequences was decided from our previous observation that substitution in the NEP SA domain of the sequence SQNS, which is polar and has alpha-helix-breaking potential, could promote SA domain processing under certain conditions (Roy, Chatellard, Lemay, Crine and Boileau (1993) J. Biol. Chem. 268. 2699-2704; Yang. Chatellard, Lazure, Crine and Boileau (1994) Arch. Biochem. Biophys. 315, 382-386). The QQTT sequence is polar but, according to secondary structure predictions, is compatible with the alpha-helix structure of the NEP SA domain. The YPGY sequence and single Pro residue are less polar and have alpha-helix-breaking potential. The predicted effects of these mutations on the structure of the NEP SA domain were confirmed by CD analysis of 42-residue peptides encompassing the hydrophobic segment and flanking regions. Wild-type and mutated proteins were expressed in COS-I cells and their fate (membrane-bound or secreted) was determined by immunoblotting and by endoglycosidase digestions. Our biochemical and structural data indicate that: (I) the cytosolic domain of NEP restricts the conformation of the SA domain because mutants not secreted in their full-length form are secreted in their truncated form; (2) alpha-helix-breaking residues are not a prerequisite for cleavage; (3) the presence, in close proximity to a putative signal peptidase cleavage site, of a polar sequence that maintains the alpha-helical structure of the SA domain is sufficient to promote cleavage. Furthermore pulse chase studies suggest that cleavage is performed in the ER by signal peptidase and indicate that cleavage is not a limiting step in the biosynthesis of the soluble form of the protein.
II型膜蛋白的信号肽/膜锚定（SA）结构域启动下游多肽跨内质网（ER）膜的转运。然而，与信号肽不同的是，SA结构域不会被信号肽酶切割，因此将蛋白质锚定在膜中。在本研究中，我们对内肽酶（中性内肽酶-24.11；NEP）的SA结构域进行了突变，以确定有利于信号肽酶对SA结构域进行加工的结构元件。通过将SQNS、QQTT或YPGY序列替换NEP SA结构域第15位起始的VTMI序列，构建了全长和截短（无细胞质结构域）蛋白的突变体。此外，在SA结构域的第16位用脯氨酸残基取代苏氨酸。使用这些序列的原理是基于我们之前的观察，即在NEP SA结构域中，具有极性且有破坏α螺旋潜力的SQNS序列的替换在某些条件下可以促进SA结构域的加工（Roy、Chatellard、Lemay、Crine和Boileau（1993）《生物化学杂志》268. 2699 - 2704；Yang、Chatellard、Lazure、Crine和Boileau（1994）《生物化学与生物物理学报》315, 382 - 386）。QQTT序列是极性的，但根据二级结构预测，与NEP SA结构域的α螺旋结构兼容。YPGY序列和单个脯氨酸残基极性较小且有破坏α螺旋的潜力。通过对包含疏水片段和侧翼区域的42个残基肽段进行圆二色性（CD）分析，证实了这些突变对NEP SA结构域结构的预测影响。野生型和突变型蛋白在COS-1细胞中表达，并通过免疫印迹和内切糖苷酶消化来确定它们的命运（膜结合或分泌）。我们的生化和结构数据表明：（I）NEP的细胞质结构域限制了SA结构域的构象，因为未以全长形式分泌的突变体以截短形式分泌；（2）破坏α螺旋的残基不是切割的先决条件；（3）在假定的信号肽酶切割位点附近存在一个保持SA结构域α螺旋结构的极性序列足以促进切割。此外，脉冲追踪研究表明切割是由信号肽酶在内质网中进行的，并表明切割不是该蛋白可溶性形式生物合成中的限制步骤。
