The YidC insertase of Escherichia coli inserts membrane proteins with small periplasmic loops (~20 residues). However, it has difficulty transporting loops that contain positively charged residues compared to negatively charged residues and, as a result, increasing the positive charge has an increased requirement for the Sec machinery as compared to negatively charged loops (Zhu et al., 2013; Soman et al., 2014). This suggested that the polarity and charge of the periplasmic regions of membrane proteins determine the YidC and Sec translocase requirements for insertion. Here we tested this polarity/charge hypothesis by showing that insertion of our model substrate protein procoat-Lep can become YidC/Sec dependent when the periplasmic loop was converted to highly polar even in the absence of any charged residues. Moreover, adding a number of hydrophobic amino acids to a highly polar loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent membrane proteins. We also demonstrate that the length of the procoat-Lep loop is indeed a determinant for Sec-dependence by inserting alanine residues that do not markedly change the overall hydrophilicity of the periplasmic loop. Taken together, the results support the polarity/charge hypothesis as a determinant for the translocase requirement for procoat insertion.

大肠杆菌的YidC插入酶插入具有小的周质环（约20个残基）的膜蛋白。然而，与带负电荷的残基相比，它难以运输包含带正电荷的残基的环，因此，与带负电荷的环相比，增加正电荷对Sec机械的要求也有所提高（Zhu等人，2013; Soman等人，2014年）。这表明，膜蛋白周质区域的极性和电荷决定了插入的YidC和Sec易位酶的要求。在这里，我们通过显示即使在没有任何带电荷残基的情况下将周质环转化为高极性时，我们的模型底物蛋白procoat-Lep的插入也可以依赖YidC / Sec来测试这种极性/电荷假设。此外，在高度极性的环中添加许多疏水性氨基酸可降低原本严格依赖Sec的膜蛋白的Sec依赖性。我们还证明，通过插入不会显着改变周质环整体亲水性的丙氨酸残基，procoat-Lep环的长度确实是Sec依赖性的决定因素。两者合计，结果支持极性/电荷假设，作为决定是否插入前涂胶的转移酶的决定因素。