Orientation of the myelin proteolipid protein C-terminus in oligodendroglial membranes.
The topology of the integral membrane proteolipid protein (PLP) has important structural and functional implications for central nervous system myelin. To determine the orientation of the carboxyl-terminal portion of PLP, cultured mouse oligodendrocytes were probed with polyclonal antibodies raised against a synthetic terminal peptide corresponding to PLP residues 264-276 and with ten separate monoclonal antibodies that react with this region. Cells were examined by double-label indirect immunofluorescence for the presence of the PLP C-terminus and either oligodendrocyte-specific surface or intracellular antigens. To detect surface antigens, both living and paraformaldehyde-fixed cells were incubated with primary antibodies and then stained with fluorochrome-conjugated second antibodies. Antigens located within the cytoplasmic space were identified after fixation and permeabilization of cells. Live-labeled oligodendrocytes were stained brightly for myelin-oligodendrocyte glycoprotein, galactocerebroside, and other surface markers but did not stain for the PLP C-terminus or the intracellular proteins myelin basic protein and beta-tubulin. Fixation alone was sufficient for partial permeabilization of oligodendrocytes to antibodies and resulted in limited staining of the PLP C-terminus and intracellular proteins. The permeabilized oligodendrocytes stained intensely for the PLP C-terminus, myelin basic protein, and beta-tubulin. Finally, trypsinization of living oligodendrocytes eliminated surface myelin-oligodendrocyte glycoprotein staining but did not change the immunostaining properties of the PLP C-terminus. These results provide evidence that the carboxyl-terminus of PLP is located at the cytoplasmic face of oligodendroglial membranes.
Konola
J T
JT
Department of Biochemistry, E.K. Shriver Center, Waltham, Massachusetts 02254.
Yamamura
T
T
Tyler
B
B
Lees
M B
MB
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000166:cytology; D002462:Cell Membrane / Q000648:ultrastructure; D002478:Cells, Cultured; D008565:Membrane Proteins / Q000648:ultrastructure*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000276:immunology / Q000648:ultrastructure*; D018991:Myelin Proteolipid Protein; D009836:Oligodendroglia / Q000737:chemistry* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000276:immunology
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D009185:Myelin Proteins; D018991:Myelin Proteolipid Protein; D010446:Peptide Fragments