Myelin/oligodendrocyte glycoprotein (MOG) is a CNS-specific integral membrane protein that is an atypical member of the immunoglobulin (Ig) superfamily with two potential transmembrane domains based upon hydropathy analysis. With only one other exception, all lg family members possess a single or no membrane spanning region. In order to analyze MOG membrane topology, we prepared stably transfected cells that express mouse MOG and used three domain-specific antisera to ascertain the localization of these hydrophilic domains. As expected, MOG's glycosylated N-terminal Ig-like domain was identified as extracellular, because membrane permeabilization was not required for immunoreactivity with the MOG 1_12, antiserum. In contrast, both MOG,54169 and MOG,95_218 antisera stained cells only upon permeabilization. These data indicate that only MOG's N-terminal hydrophobic domain spans the lipid bilayer, and we propose that MOG's C-terminal hydrophobic domain associates with the cytoplasmic face of the plasma membrane. As for MOG's second hydrophobic domain, it is clear that either orientation (transmembrane versus membrane-associated) would be unique among Ig-like proteins, and the implications of our proposed topology for MOG in oligodendroglial plasma membrane are discussed.

Oligodendrocytes possess two distinct membrane compartments ± uncompacted plasma membrane (cell body, processes) and compact myelin. Speci®c targeting mechanisms must exist to establish and maintain these membrane domains. Polarized epithelial cells have the best characterized system for targeting components to apical and basolateral compartments. Since oligodendrocytes arise from neuroepithelial cells, we investigated whether they might utilize targeting paradigms similar to polarized epithelial cells. Myelin/oligodendrocyte glycoprotein (MOG) is a transmembrane Ig-like molecule restricted to uncompacted oligodendroglial plasma membrane. We stably expressed MOG in Madin±Darby canine kidney (MDCK) Type II epithelial cells, which have been extensively used in protein-targeting studies. Data from surface biotinylation assays and confocal microscopy revealed that MOG sorts exclusively to the basolateral membrane of MDCK cells. Expression vectors containing progressive truncations of MOG from the cytoplasmic C-terminus were expressed in MDCK cells to localize basolateral sorting signals. A loss of only four C-terminal residues results in some MOG expression at the apical surface. More strikingly, removal of the C-terminal membrane associated hydrophobic domain from MOG results in complete loss of basolateral sorting and speci®c targeting to the apical membrane. These data suggest that myelinating oligodendrocytes may utilize a sorting mechanism similar to that of polarized epithelia. Keywords: basolateral sorting, cell polarity, immunoglobulin superfamily, membrane targeting, multiple sclerosis. A myelinating oligodendrocyte elaborates a complex array of cellular architectures with numerous slender processes that project outward from its cell body to terminate in highly specialized membrane sheets of compact myelin. While the cytoplasmic compartment is contiguous from the cell body to the myelin sheath, distinct membrane domains are evident with clear asymmetric regional localizations of lipids and proteins. As compared with membranes of the cell body and processes, compact myelin is particularly enriched in glycosphingolipids and cholesterol (Norton and Cammer 1984;Morell and Quarles 1999). Moreover, speci®c membrane proteins show restricted expression patterns in periaxonal areas (i.e. myelin-associated glycoprotein, MAG), compact myelin (i.e. proteolipid protein, PLP), or at the oligodendrocyte cell body (i.e. myelin/oligodendrocyte glycoprotein, MOG) (Eng et al. 1968;Webster et al. 1983;Brunner et al. 1989).MOG is a CNS-speci®c integral membrane protein that is localized to oligodendrocyte cell bodies, oligodendroglial processes and the outermost wraps of myelin sheaths; it is virtually excluded from compact myelin layers and the periaxonal space (Linington et al. 1988;Brunner et al. 1989). A consideration of the asymmetric distribution of MOG in oligodendrocytes led us to study its membrane targeting. It was originally identi®ed by a mouse monoclonal

Oligodendrocytes elaborate an extensive membrane network that ensheathes CNS axons in multilamellar wrappings. A compaction process excludes much of the cytoplasm in mature myelin membranes, giving rise to distinct lipid/protein compositions in two membrane compartments (compact myelin and membranes of the cell body and processes). Insofar as oligodendrocytes arise from neuroepithelial progenitors, it seems likely that some elements are shared for protein targeting by these two cell types. We hypothesized that certain membrane proteins targeting different oligodendroglial membrane compartments would preferentially sort to opposite domains when transfected into Madin-Darby canine kidney (MDCK) epithelial cells. Myelin/oligodendrocyte glycoprotein (MOG) is found in uncompacted membrane (cell body, processes), and it sorts exclusively to MDCK basolateral membrane. Proteolipid protein (PLP) is found in compact myelin, and it sorts exclusively to MDCK apical membrane. Myelin-associated glycoprotein (MAG) is primarily in the periaxonal inner loop of myelin; however, it fails to target preferentially within MDCK cells. This inability of MAG to sort within MDCK cells suggests a lack of required oligodendroglial-specific targeting components. In contrast, the sorting machinery in both oligodendrocytes and MDCK cells recognizes targeting signals for MOG and PLP, and we propose that these oligodendroglial membrane proteins delineate cognate basolateral and apical domains, respectively.

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