Xie Kun, Hessa Tara, Seppälä Susanna, Rapp Mikaela, von Heijne Gunnar, Dalbey Ross E
Department of Chemistry, The Ohio State University, 100 W. 18th Avenue, Columbus, Ohio 43210, USA.
Biochemistry. 2007 Dec 25;46(51):15153-61. doi: 10.1021/bi701398y. Epub 2007 Dec 4.
Topogenic sequences direct the membrane topology of proteins by being recognized and decoded by integral membrane translocases. In this paper, we have compared the minimal sequence characteristics of helical-hairpin, reverse signal-anchor, and stop-transfer sequences in bacterial membrane proteins that use either the YidC or SecYEG translocases for membrane insertion. We find that a stretch composed of 3 leucines and 16 alanines is required for efficient membrane-anchoring of the M13 procoat protein that inserts by a helical hairpin mechanism, and that a stretch composed of only 19 alanines has a detectable membrane-anchoring ability. Similar results were obtained for the reverse signal-anchor sequence of the single-spanning Pf3 coat protein and for stop-transfer segments engineered into leader peptidase. We have also determined the contribution to the apparent free energy of membrane insertion of M13 procoat for all 20 amino acids. The relative order of the contributions is similar to that determined for a stop-transfer sequence in the mammalian endoplasmic reticulum, but the absolute difference between the contributions for the most hydrophobic and most hydrophilic residues is somewhat larger in the E. coli system. These results are significant because they define the features of a membrane protein transmembrane segment that induce lateral release from the YidC and Sec translocases into the lipid bilayer in bacteria.
拓扑序列通过被整合膜转运酶识别和解码来指导蛋白质的膜拓扑结构。在本文中，我们比较了利用YidC或SecYEG转运酶进行膜插入的细菌膜蛋白中螺旋发夹、反向信号锚定和终止转移序列的最小序列特征。我们发现，通过螺旋发夹机制插入的M13原衣壳蛋白有效膜锚定需要一段由3个亮氨酸和16个丙氨酸组成的序列，而仅由19个丙氨酸组成的一段序列具有可检测到的膜锚定能力。对于单跨Pf3衣壳蛋白的反向信号锚定序列以及工程改造到前导肽酶中的终止转移片段，也获得了类似的结果。我们还确定了所有20种氨基酸对M13原衣壳膜插入表观自由能的贡献。贡献的相对顺序与哺乳动物内质网中终止转移序列所确定的顺序相似，但在大肠杆菌系统中，最疏水和最亲水残基贡献之间的绝对差异略大。这些结果意义重大，因为它们定义了细菌中膜蛋白跨膜片段从YidC和Sec转运酶侧向释放到脂质双层中的特征。
