Nothwehr S F, Hoeltzli S D, Allen K L, Lively M O, Gordon J I
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1990 Dec 15;265(35):21797-803.
The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation. To identify structural features of residues flanking the c-region that influence the fidelity and efficiency of signal peptidase cleavage as well as co-translational translocation, we introduced six amino acid substitutions into the COOH terminus of the hydrophobic core and seven substitutions at the NH2 terminus of the mature region (the +1 position) of a model eukaryotic preprotein-human pre(delta pro)apoA-II. This preprotein contains several potential sites for signal peptidase cleavage. The functional consequences of these mutations were assayed using an in vitro co-translational translocation/processing system and by post-translational cleavage with purified, detergent-solubilized, hen oviduct signal peptidase. The efficiency of translocation could be correlated with the hydrophobic character of the residue introduced at the COOH terminus of the h-region. Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage. These data suggest that features at the COOH terminus of the h-domain can influence "presentation" of the cleavage site to signal peptidase. The +1 residue substitutions had minor effects on the extent of co-translational translocation and processing. However, these +1, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing. The site(s) selected by canine microsomal and purified hen oviduct signal peptidase were very similar, suggesting that "intrinsic" structural features of this prepeptide can influence the selectivity of eukaryotic signal peptidase cleavage, independent of the microsomal membrane and associated translocation apparatus.
信号肽的极性COOH末端c区域被认为对影响信号肽酶切割的效率和准确性最为重要，而疏水核心或h区域似乎对启动转运是必不可少的。为了确定影响信号肽酶切割的准确性和效率以及共翻译转运的c区域侧翼残基的结构特征，我们在模型真核前体蛋白——人前（δpro）载脂蛋白A-II的疏水核心的COOH末端引入了六个氨基酸替换，并在成熟区域的NH2末端（+1位置）引入了七个替换。该前体蛋白包含几个信号肽酶切割的潜在位点。使用体外共翻译转运/加工系统以及用纯化的、去污剂溶解的鸡输卵管信号肽酶进行翻译后切割来检测这些突变的功能后果。转运效率可能与在h区域COOH末端引入的残基的疏水特性相关。一些h/c边界突变体在微粒体膜上进行共翻译转运时仅有最小程度的切割，但它们被鸡输卵管信号肽酶进行翻译后切割的效率比其他表现出共翻译转运和切割高度偶联的突变体更高。这些数据表明h结构域COOH末端的特征可以影响切割位点向信号肽酶的“呈现”。+1残基替换对共翻译转运和加工的程度影响较小。然而，这些+1以及h/c边界突变对信号肽酶选择的切割位点有显著影响，表明该原型真核信号肽c区域侧翼的残基可以影响其蛋白水解加工的准确性。犬微粒体和纯化的鸡输卵管信号肽酶选择的位点非常相似，表明该前肽的“内在”结构特征可以影响真核信号肽酶切割的选择性，而与微粒体膜和相关的转运装置无关。
