Noda Y, Yoda K, Takatsuki A, Yamasaki M
Department of Agricultural Chemistry, University of Tokyo, Japan.
J Bacteriol. 1992 Jul;174(13):4302-7. doi: 10.1128/jb.174.13.4302-4307.1992.
tmrB is the gene responsible for tunicamycin resistance in Bacillus subtilis. It is predicted that an increase in tmrB gene expression makes B. subtilis tunicamycin resistant. To examine the tmrB gene product, we produced the tmrB gene product in Escherichia coli by using the tac promoter. TmrB protein was found not only in the cytoplasm fraction but also in the membrane fraction. Although TmrB protein is entirely hydrophilic and has no hydrophobic stretch of amino acids sufficient to span the membrane, its C-terminal 18 amino acids could form an amphiphilic alpha-helix. Breaking this potential alpha-helix by introducing proline residues or a stop codon into this region caused the release of this membrane-bound protein into the cytoplasmic fraction, indicating that the C-terminal 18 residues were essential for membrane binding. On the other hand, TmrB protein has an ATP-binding consensus sequence in the N-terminal region. We have tested whether this sequence actually has the ability to bind ATP by photoaffinity cross-linking with azido-[alpha-32P]ATP. Wild-type protein bound azido-ATP well, but mutants with substitutions in the consensus amino acids were unable to bind azido-ATP. These C-terminal or N-terminal mutant genes were unable to confer tunicamycin resistance on B. subtilis in a multicopy state. It is concluded that TmrB protein is a novel ATP-binding protein which is anchored to the membrane with its C-terminal amphiphilic alpha-helix.
tmrB是负责枯草芽孢杆菌对衣霉素耐药性的基因。据预测，tmrB基因表达的增加会使枯草芽孢杆菌对衣霉素产生耐药性。为了研究tmrB基因产物，我们利用tac启动子在大肠杆菌中生产了tmrB基因产物。发现TmrB蛋白不仅存在于细胞质部分，也存在于膜部分。虽然TmrB蛋白完全亲水，没有足够长的疏水氨基酸序列来跨越膜，但它的C末端18个氨基酸可以形成两亲性α-螺旋。通过在该区域引入脯氨酸残基或终止密码子来破坏这种潜在的α-螺旋，会导致这种膜结合蛋白释放到细胞质部分，这表明C末端的18个残基对于膜结合至关重要。另一方面，TmrB蛋白在N末端区域有一个ATP结合共有序列。我们通过与叠氮基-[α-32P]ATP进行光亲和交联，测试了该序列是否真的具有结合ATP的能力。野生型蛋白能很好地结合叠氮基ATP，但在共有氨基酸处有取代的突变体则无法结合叠氮基ATP。这些C末端或N末端突变基因在多拷贝状态下无法赋予枯草芽孢杆菌对衣霉素的耐药性。得出的结论是，TmrB蛋白是一种新型的ATP结合蛋白，其通过C末端两亲性α-螺旋锚定在膜上。
