INS AND OUTS OF PERIPHERAL MYELIN PROTEIN-22 - MAPPING TRANSMEMBRANE TOPOLOGY AND INTRACELLULAR SORTING

Molecular genetic studies have shown that the peripheral myelin protei n 22 (PMP22) is a key gene in hereditary peripheral neuropathies and a ppears to be essential for the formation and maintenance of myelin in the PNS. Based on the amino acid sequence the predicted structure of P MP22 protein contains two major distinct hydrophilic regions and four transmembrane domains. To analyze the cellular localization and membra ne topology of PMP22 we inserted an octapeptide tag-sequence at the am ino or at the carboxyl terminus of the PMP22 open reading frame and ge nerated different chimeric constructs which were expressed in HeLa cel ls. The expression of the tagged PMP22 protein and its orientation wit h respect to the plasma membrane were analyzed using antibodies raised against specific PMP22 epitopes and the tag sequence. Combined indire ct, double-immunofluorescence labeling and confocal microscopy showed that PMP22 is synthesized in the rough endoplasmic reticulum of transf ected cells and passes through the Golgi apparatus to the cell surface . We determined the transmembrane organization of PMP22 providing the first experimental evidence that confirms the cytoplasmic disposition of its N and C termini and the extracellular localization of the two h ydrophilic domains containing amino acids 28-40 and 118-131. This stud y provides the basis for further analysis aimed to identify functional domains of wild-type PMP22 and the cellular sorting of mutant forms o f PMP22. (C) 1997 Wiley-Liss, Inc.