Peripheral myelin protein 22 (PMP22) is an integral membrane protein that is essential for the normal formation and maintenance of peripheral myelin. Duplications, deletions, or mutations in the PMP22 gene account for a set of dominantly inherited peripheral neuropathies. The heterozygous Trembler-J (TrJ) genotype in mice is similar genetically to a Charcot-Marie-Tooth disease type 1A pedigree in humans, whereas the homozygous TrJ condition leads to the most severe form of PMP22-associated neuropathies. To characterize the consequences of the TrJ mutation, we labeled wild-type (wt-) and TrJ-PMP22 in the third loop of the protein with different epitope tags and expressed them separately or together in COS7 cells and primary Schwann cells. Here we show that the transport of the mutant TrJ-PMP22 is interrupted in the intermediate compartment, preventing its insertion into the plasma membrane and affecting the morphology of the endoplasmic reticulum. In addition, TrJ-PMP22 forms a heterodimer with the wt-PMP22. This interaction causes a fraction of the wt-PMP22 to be retained with TrJ-PMP22 in the intermediate compartment of COS7 and Schwann cells. The relative stability of a wt-mutant PMP22 heterodimer as compared with the wt-wt PMP22 homodimer may determine whether a particular mutation is semidominant or dominant. The neuropathy itself appears to result both from decreased trafficking of wt-PMP22 to the plasma membrane and from a toxic gain of function via the accumulation of wt- and TrJ-PMP22 in the intermediate compartment.

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Mutations in the gene encoding the peripheral myelin protein 22 (PMP22), a tetraspan protein in compact peripheral myelin, are one of the causes of inherited demyelinating peripheral neuropathy. Most PMP22 mutations alter the trafficking of the PMP22 protein in Schwann cells, and this different trafficking has been proposed as the underlying mechanism of the disease. To explore this problem further, we compared the aggregation of wild-type Pmp22 with those of the two Pmp22 mutations found in Trembler (Tr) and Trembler J (TrJ) mice. All three Pmp22s can be crosslinked readily as homodimers in transfected cells. Wild-type Pmp22 also forms heterodimers with Tr and TrJ Pmp22, and these heterodimers traffic with their respective mutant Pmp22 homodimers. All three Pmp22s form complexes larger than dimers with Tr Pmp22 especially prone to aggregate into high molecular weight complexes. Despite the differences in aggregation of Tr and TrJ Pmp22, these two mutant Pmp22s sequester the same amount of wild-type Pmp22 in heterodimers and heterooligomers. Thus, the differences in the phenotypes of Tr and TrJ mice may depend more on the ability of the mutant protein to aggregate than on the dominant-negative effect of the mutant Pmp22 on wild-type Pmp22 trafficking.

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Octamer transcription factors (Oct or OTF) are a subset of the POU family of transcription factors which regulate transcription of cellular and viral genes by binding to the octamer sequence motif ATGCAAAT. Neurons and astroglial cells harbour, in addition to the ubiquitous Oct 1 factor, at least four specific factors termed N-Oct 2,3,4 and 5. Here we report the cloning of a human brain-derived cDNA that encodes the N-Oct 3 protein (443 aa) which is the human counterpart of the murine brain-2 gene product. Extracts from mammalian cells transfected with an N-Oct 3 expression vector yield three octamer DNA binding complexes in the electrophoretic mobility shift assay (EMSA): N-Oct 3 and two smaller complexes comigrating with the N-Oct 5A and 5B proteins of brain extracts. We present data suggesting that the N-Oct 5A and 5B proteins are generated by alternative translation initiation at internal AUG residues which are located before the POU domain. In contrast to the putative N-Oct 5 proteins, which are transcriptionally inert, the N-Oct 3 protein activates transcription from a reporter gene promoter with an octamer sequence, when transiently expressed in HeLa cells.