Department Biochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Universitätsstr. 150, 44801, Bochum, Germany.
Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, Albert-Ludwigs-University Freiburg, Freiburg im Breisgau, Germany.
Sci Rep. 2020 Oct 1;10(1):16347. doi: 10.1038/s41598-020-73185-y.
OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, which is critical to regulate pore selectivity, is also required for SecY-mediated transport of OmpC and OmpF into the periplasm. Variants with a deleted or mutated motif were expressed in the cytosol and translocation was initiated. However, after signal peptide cleavage, import into the periplasm was aborted and the mutated proteins were redirected to the cytosol. Strikingly, reducing the proof-reading capacity of SecY by introducing the PrlA4 substitutions restored transport of OmpC with a mutated channel domain into the periplasm. Our study identified a SecY-mediated quality control pathway to restrict transport of outer membrane porin proteins with a deregulated channel activity into the periplasm.
OmpC 和 OmpF 是大肠杆菌中最丰富的外膜蛋白之一，它们作为亲水性通道，介导包括抗生素在内的小分子的摄取。流入的选择性由通道的所谓收缩区或孔眼控制。形成孔眼的环域中的突变会破坏运输的选择性，从而干扰细菌的存活能力。在这项研究中，我们表明，孔眼中一个高度保守的、由五个带负电荷的氨基酸组成的模体对于调节孔选择性至关重要，对于 SecY 介导的 OmpC 和 OmpF 向周质的转运也是必需的。缺失或突变模体的变体在细胞质中表达，并启动了易位。然而，在信号肽切割后，导入周质的过程被中止，突变蛋白被重新导向细胞质。引人注目的是，通过引入 PrlA4 取代物降低 SecY 的校对能力，恢复了突变通道结构域的 OmpC 向周质的转运。我们的研究确定了一种 SecY 介导的质量控制途径，以限制具有失调通道活性的外膜孔蛋白向周质的转运。
