Lewis G Shane, Jewell Jean E, Phang Tzulip, Miller Kurt W
Department of Molecular Biology, University of Wyoming, P.O. Box 3944, Laramie, WY 82071-3944, USA.
Arch Biochem Biophys. 2002 Aug 15;404(2):317-25. doi: 10.1016/s0003-9861(02)00287-4.
The tetracycline resistance proteins (TetA) of gram-negative bacteria are secondary active transport proteins that contain buried charged amino acids that are important for tetracycline transport. Earlier studies have shown that insertion of TetA proteins into the cytoplasmic membrane is mediated by helical hairpin pairs of transmembrane (TM) segments. However, whether helical hairpins direct spontaneous insertion of TetA or are required instead for its interaction with the cellular secretion (Sec) machinery is unknown. To gain insight into how TetA proteins are inserted into the membrane, we have investigated how tolerant the class C TetA protein encoded by plasmid pBR322 is to placement of charged residues in TM segments. The results show that the great majority of charge substitutions do not interfere with insertion even when placed at locations that cannot be shielded internally within helical hairpins. The only mutations that frequently block insertion are proline substitutions, which may interfere with helical hairpin folding. The ability of TetA to broadly tolerate charge substitutions indicates that the Sec machinery assists in its insertion into the membrane. The results also demonstrate that it is feasible to engineer charged residues into the interior of TetA proteins for the purpose of structure-function analysis.
革兰氏阴性菌的四环素抗性蛋白（TetA）是次级主动转运蛋白，其含有对四环素转运很重要的埋藏的带电荷氨基酸。早期研究表明，TetA蛋白插入细胞质膜是由跨膜（TM）片段的螺旋发夹对介导的。然而，螺旋发夹是直接引导TetA的自发插入，还是其与细胞分泌（Sec）机制相互作用所必需的，目前尚不清楚。为了深入了解TetA蛋白如何插入膜中，我们研究了质粒pBR322编码的C类TetA蛋白对TM片段中带电荷残基位置的耐受性。结果表明，即使将绝大多数电荷替代置于螺旋发夹内部无法屏蔽的位置，也不会干扰插入。经常阻断插入的唯一突变是脯氨酸替代，这可能会干扰螺旋发夹折叠。TetA广泛耐受电荷替代的能力表明Sec机制有助于其插入膜中。结果还表明，为了结构 - 功能分析的目的，将带电荷残基工程化到TetA蛋白内部是可行的。
