Organelle targeting and folding of membrane proteins

本研究以膜拓扑发生和分选为研究背景，对内质网膜上的拓扑发生过程进行了广泛的研究，并取得了以下进展。(1)I型信号锚定序列可使N端部分易位，从而介导含有DHFR结构域的N结构域的易位。通过使用DHFR配体MTX，可以调节N-末端DHFR结构域的易位。对于长N-结构域的易位，既不需要NTP也不需要管腔hsp 70同源物BiP。然而，核糖体在N结构域移位中起着关键作用。在初始阶段，N-结构域的驱动力远大于后一阶段。(2)NHX 1是NHE基因家族的植物同源物，具有与动物相同的膜拓扑结构，表明NHE基因家族成员的基本拓扑结构。(3)在内质网上膜蛋白的拓扑发生中，疏水跨膜片段的方向受到侧翼氨基酸残基的电荷的影响。在多肽延伸过程中电荷的作用时间表明，这些功能在核糖体出口位点，并且疏水片段向特定方向的承诺受到多肽链远下游部分的影响。(4)我们开发了一种新型的可调控的N结构域易位实验系统。在多跨膜拓扑发生过程中，单个膜蛋白中的两个易位亲水片段可以跨膜，位于易位子两侧。此外，即使在六个连续的疏水片段进入易位子，N-结构域易位可以诱导重新启动从一个被捕的状态。这表明了易位子的显著灵活性。

In this research, based on our research background of membrane topogenesis and sorting, we have extensively examined topogenesis process on the endoplasmic reticulum membranes and obtained the following progress. (1) The type I signal-anchor sequence, which translocates the N-terminal portion, can mediate the translocation of N-domain containing DHFR-domain. By using DHFR ligand, MTX, the N-terminal DHFR domain translocation can be regulated. For the translocation of the long N-domain, neither NTP's nor luminal hsp70 homologue, BiP, is required. Ribosome, however, plays a critical function for the N-domain translocation. The driving force for the N-domain at the initial stage is much larger than that of the latter stage. (2) The plant homologue of NHE gene family, NHX1 possesses the same membrane topology as that of animal, indicating the fundamental topology of the NHE gene family members. (3) In topogenesis of membrane proteins on the endoplasmic reticulum, the orientation of the hydrophobic transmembrane segment is influenced by the charge of the flanking amino acid residues. The timing of action of the charges during polypeptide elongation indicated that those function at the ribosome exit sites and the commitment of the hydrophobic segment to a particular orientation is influenced by far downstream parts of the polypeptide chain. (4) We developed novel regulatable experiment system for N-domain translocation. Two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. The remarkably flexible nature of the translocon was indicated.