Akiyama Y, Ito K
Institute for Virus Research, Kyoto University, Japan.
J Biol Chem. 1989 Jan 5;264(1):437-42.
The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides.
SecY蛋白是细菌蛋白质输出所必需的一种膜结合因子，通过其10个跨膜区段嵌入细胞质膜中。我们之前通过采用Manoil-Beckwith TnphoA方法（一种根据连接到不同区域的碱性磷酸酶（PhoA）成熟序列的酶活性来确定膜蛋白局部定位的遗传方法）为该蛋白提出了一种拓扑模型。在SecY序列的五个假定周质结构域处获得了PhoA结构域输出到膜周质侧的SecY-PhoA杂合蛋白。我们现在扩展了这种方法，将其应用于追踪新合成的PhoA结构域的输出。用胰蛋白酶处理去污剂溶解的细胞提取物会消化内化的（未折叠的）PhoA结构域，但不会消化输出并正确折叠的结构域。其中一种杂合蛋白在输出到周质后在体内被切割，为输出提供了便利的指示。这些分析结果表明，连接到SecY不同周质区域的PhoA结构域的输出迅速发生，并且需要反式提供的secY基因的正常功能。因此，这种具有多个跨膜区段的膜蛋白包含多个输出信号，这些信号可以促进连接到羧基末端侧的PhoA成熟序列的快速且依赖secY的输出。
