Strauch K L, Beckwith J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1988 Mar;85(5):1576-80. doi: 10.1073/pnas.85.5.1576.
A fusion between tsr (encoding the inner membrane protein Tsr) and phoA (encoding the periplasmic protein alkaline phosphatase, AP) generates a membrane-bound hybrid protein (Tsr-AP 2) with AP enzymatic activity. The hybrid protein is proteolytically unstable and is broken down to yield a smaller, soluble species with AP activity. We devised a genetic screen to distinguish between cells containing only membrane-bound AP and those containing soluble AP. The screen depends on diffusion of soluble AP away from cells with a leaky outer membrane to produce a halo of AP activity around colonies on solid growth medium. Several mutants lacking this halo show reduced degradation of Tsr-AP 2. One mutant is also defective in breakdown of five other abnormal periplasmic proteins but not of two cytoplasmic proteins. The mutation in this strain, degP4::Tn5, defines a locus distinct from previously identified loci that affect protein stability or protease activities. This strain may be useful for preventing the breakdown of unstable foreign proteins in Escherichia coli.
tsr（编码内膜蛋白Tsr）与phoA（编码周质蛋白碱性磷酸酶，AP）融合产生具有AP酶活性的膜结合杂合蛋白（Tsr-AP 2）。该杂合蛋白在蛋白水解方面不稳定，会被分解产生一种更小的、具有AP活性的可溶性物质。我们设计了一种遗传筛选方法，以区分仅含有膜结合AP的细胞和含有可溶性AP的细胞。该筛选方法依赖于可溶性AP从外膜有渗漏的细胞扩散开来，从而在固体生长培养基上的菌落周围产生AP活性晕圈。几个缺乏这种晕圈的突变体显示Tsr-AP 2的降解减少。一个突变体在分解其他五种异常周质蛋白时也有缺陷，但对两种细胞质蛋白没有影响。该菌株中的突变，degP4::Tn5，定义了一个与先前鉴定的影响蛋白质稳定性或蛋白酶活性的基因座不同的基因座。该菌株可能有助于防止大肠杆菌中不稳定的外源蛋白被分解。
