Wahle S, Stoffel W
Institute of Biochemistry, Faculty of Medicine, University of Cologne, Germany.
Glia. 1998 Oct;24(2):226-35. doi: 10.1002/(sici)1098-1136(199810)24:2<226::aid-glia7>3.0.co;2-1.
The four transmembrane domain topology of the proteolipid protein (PLP) in the myelin membrane of the central nervous system (CNS) has been further substantiated by biochemical studies. We have analyzed the cotranslational polytopic integration of nascent PLP during protein synthesis into the membrane of the endoplasmic reticulum (ER) on two routes. Consensus sequences for N-glycosylation were introduced by site directed mutagenesis into the PLP sequence as reporter sites, which upon glycosylation monitor the intraluminal location of the respective domains corresponding to the extracellular side of the plasma membrane. Single, double, and triple mutant cDNAs were constructed for transcription/translation in vitro in the presence of ER-membranes. The glycosylation pattern of the translation products revealed that hydrophilic extramembrane regions 2 and 4 (EMR2/EMR4) and EMR3 of PLP are exposed on opposite sides of the ER membrane. Their localization either at the cytosolic or luminal side of the ER membrane leads to two different topologies. The two modes of membrane integration during in vitro cotranslational translocation were confirmed by protease protection assays with wild-type and truncated PLP polypeptides with either one, two, or three putative transmembrane domains integrated into the ER-membrane. The fragment pattern of the [35S]methionine- or [3H]leucine-labeled polypeptides revealed that EMR3 and EMR4 were exposed with opposite orientation either on the cytosolic or luminal side of the ER membrane supporting the 4-transmembrane helix (TMH) N(in) model with the N and C termini on the cytoplasmic side, as established for the myelin membrane (plasma membrane); the other inversely integrated PLP constructs indicate the 4-TMH-Nout profile. These results are discussed with regard to the PLP biogenesis and the plasma membrane topology in PLP-expressing cells.
中枢神经系统（CNS）髓鞘膜中蛋白脂蛋白（PLP）的四跨膜结构域拓扑结构已通过生化研究得到进一步证实。我们分析了新生PLP在蛋白质合成过程中通过两条途径共翻译多跨膜整合到内质网（ER）膜中的情况。通过定点诱变将N-糖基化的共有序列引入PLP序列作为报告位点，糖基化时可监测对应于质膜胞外侧的各个结构域在腔内的位置。构建了单突变、双突变和三突变cDNA，用于在ER膜存在的情况下进行体外转录/翻译。翻译产物的糖基化模式显示，PLP的亲水性膜外区域2和4（EMR2/EMR4）以及EMR3暴露于ER膜的相对两侧。它们定位于ER膜的胞质侧或腔侧会导致两种不同的拓扑结构。通过用野生型和截短的PLP多肽进行蛋白酶保护试验，证实了体外共翻译转运过程中的两种膜整合模式，这些多肽带有一个、两个或三个假定的跨膜结构域整合到ER膜中。[35S]甲硫氨酸或[3H]亮氨酸标记的多肽的片段模式显示，EMR3和EMR4以相反的方向暴露于ER膜的胞质侧或腔侧，支持四跨膜螺旋（TMH）N（in）模型，其N端和C端位于细胞质侧，这已在髓鞘膜（质膜）中得到证实；其他反向整合的PLP构建体则显示出四跨膜螺旋N（out）模式。本文就PLP生物发生和表达PLP的细胞中的质膜拓扑结构对这些结果进行了讨论。
