| The precise functions of the major myelin proteins remain unknown, even after much research in the field. The work presented herein investigates myelin protein function by identifying protein-binding partners and by co-localizing proteins to isolated myelin membrane microdomains. Towards this goal, co-immunoprecipitation analysis of the Triton-soluble extract revealed that a portion of the major myelin proteins MBP, PLP and the small isoform of MAG (S-MAG) formed a complex in myelin. The PLP-MBP-S-MAG complex also contained p42 MAPK, some phosphorylated proteins and likely occurs in the periaxonal region or paranodal loops of myelin. Flotation of the Triton-insoluble extract on sucrose density gradients revealed two low-density fractions (B1 and B2), both indicative of lipid rafts since they were enriched in GalC, cholesterol, and contained the GPI-linked NCAM120. The rafts had size-isoform specificity for PLP, MAG, and MAPK, while actin and tubulin were localized primarily to B2. The differential protein composition of B1 and B2 suggests distinct functions of these two myelin microdomains. Furthermore, the characterization of myelin extracts led to the identification of MEK and CD44, which had not been previously shown to be in myelin. Also, an ER similar to ERbeta and caveolin-1 were identified in myelin membranes by 2D-PAGE, mass spectrometry and western blotting. These proteins had also not been previously shown to occur in myelin. Knowledge of the function of myelin protein interactions is central to understanding myelin formation, compaction, maintenance and dynamic functions such as communication with the axon. |
