Kinetic analysis of the assembly of the outer membrane protein LamB in Escherichia coli mutants each lacking a secretion or targeting factor in a different cellular compartment
Kinetic analysis of the assembly of the outer membrane protein LamB in Escherichia coli mutants each lacking a secretion or targeting factor in a different cellular compartment
复制标题
DOI：
10.1128/jb.01103-06
复制
发表时间：
2007-01-01
影响因子：
3.2
通讯作者：
Silhavy, Thomas J.
中科院分区：
文献类型：
--
作者：
Ureta, Alejandro R.;Endres, Robert G.;Silhavy, Thomas J.
革兰氏阴性细菌（例如大肠杆菌）中的外膜 P 桶蛋白必须从细胞质中的合成位点转移到周质，并最终传递到外膜。催化这一复杂的组装过程需要至少十几种位于细胞质、周质以及内膜和外膜中的蛋白质。在正常的生长温度和条件下，运输和组装过程非常快，以至于无法检测到组装中间体。利用低温生长的细胞来减缓组装过程，并通过免疫检测方法进行脉冲追踪分析，我们在新合成的 LamB 分子穿过细胞包膜的过程中对其进行跟踪。数据的质量和再现性使我们能够计算三种不同组件反应的速率常数。该动力学分析表明，secB 和 secD 突变体在从细胞质的前体易位方面表现出几乎相同的缺陷。然而，随后的子组装反应速率没有提供明确的证据来证明 SeeD 在 LamB 组装中的额外作用。此外，我们发现 surA 突变体在质量上与 yfgL 突变体没有区别，这表明这两个基因的产物在组装过程中具有共同的功能，很可能将 LamB 传递到外膜中的 YaeT 组装复合物。
Outer membrane P-barrel proteins in gram-negative bacteria, such as Escherichia coli, must be translocated from their site of synthesis in the cytoplasm to the periplasm and finally delivered to the outer membrane. At least a dozen proteins located in the cytoplasm, the periplasm, and both the inner and outer membranes are required to catalyze this complex assembly process. At normal growth temperatures and conditions the transport and assembly processes are so fast that assembly intermediates cannot be detected. Using cells grown at a low temperature to slow the assembly process and pulse-chase analysis with immunodetection methods, we followed newly synthesized LamB molecules during their transit through the cell envelope. The quality and reproducibility of the data allowed us to calculate rate constants for three different subassembly reactions. This kinetic analysis revealed that secB and secD mutants exhibit nearly identical defects in precursor translocation from the cytoplasm. However, subsequent subassembly reaction rates provided no clear evidence for an additional role for SeeD in LamB assembly. Moreover, we found that surA mutants are qualitatively indistinguishable from yfgL mutants, suggesting that the products of both of these genes share a common function in the assembly process, most likely the delivery of LamB to the YaeT assembly complex in the outer membrane.