Dissection of polypeptide translocator function of SecY

SECY是一种完整的膜蛋白，被认为是大肠杆菌中蛋白质转运体的中心成分。我们已经确定了SECY的显性负突变，其表达干扰了蛋白质的输出。这些突变被证明存在于SecY的C-末端三分之一，主要位于细胞质结构域5(C5)。其中C5-TM9界面的三个残基缺失(Secy^D1)为强显性-负性。占主导地位的负性突变蛋白将隔离非活性复合体中相互作用的成分，从而与SecY^+竞争形成功能性SecY复合体。与我们的模型一致的是，C4区的SecY24(Ts)突变被发现削弱了SecY与SecE的相互作用。此外，secDF操纵子中SecE或“ORF12”的过量生产克服了出口干扰。此外，我们还发现了一个新的基因YDR，它是Secy^D1的另一个剂量依赖的克服者。虽然SecY的过度表达(可能没有复杂)在体内迅速降解，但同时过量生产SecE、Orf12或YDR显著稳定了它。有趣的是，在secY24突变细胞中YDR的过量生产严重干扰了蛋白质的输出和活性。YDR是一种具有181个残基的亲水性蛋白质，其C末端可能形成一个两亲性的α-螺旋，它与膜的结合是松散的。最后，我们讨论了膜蛋白整合是否需要任何因素。我们的突变分析表明，FtsH是一个与真核细胞ATPase家族同源的膜结合ATPase，它在确保疏水伸展有效地锚定到膜上以及转位过程本身中发挥着作用。

SecY is an integral membrane protein and believed to be a central component of the protein translocator in Escherichia coli. We have identified dominant negative mutations of secY whose expression interfered with protein export. Such mutations proved to reside in the C-terminal third of SecY, mostly in the cytoplasmic domain 5 (C5). On of them (secY^d1), a three residue deletion in the C5-TM9 interface, was strongly dominant-negative. The dominant negative mutant protein will sequester the interacting components in an inactive complex, and hence compete with SecY^+ for the formtion of the functiona SecY complex. Linker insertion mutations that suppressed secY^d1 were localized within the C4-TM7 region is important for SecY's interaction with other components of the system.Consistent with our model, the secY24(Ts) mutation in the C4 domain was found to impair the SecY's interaction with SecE. Also, overproduction of either SecE or "ORF12" in the secDF operon overcame the export interference. Futhermore, we were able to identify a new gene, ydr, as another dosage-dependent overcomer of secY^d1. Although overexpressed (and presumably uncomplexed) SecY was rapidly degraded in vivo, it was significantly stabilized by simultaneous overproduction of either SecE, Orf12,or Ydr. Interestingly, overproduction of Ydr in the secY24 mutant cell severely interfered with protein export and the viability. Ydr, a hydrophilic 181-residue protein with is C-terminal region potentially forming an amphiphilic alpha-helix, is loosely associated with the membrane.Finally we addressed whether any factors are required for membrane protein integration. Our mutational analyses suggested that FtsH, a putative membrane bound ATPase with homology to a family of eukaryotic ATPases, has a role in assuring efficient anchoring of a hydrophobic stretch to the membrane as well as in the translocation process itself.