Cotranslational integration of myelin proteolipid protein (PLP) into the membrane of endoplasmic reticulum: analysis of topology by glycosylation scanning and protease domain protection assay.

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.