Our long range goal is to understand the molecular mechanisms that underlie the formation of the mature myelin sheath, in the realization that this may suggest ways by which the repair process can be encouraged after myelin destruction. An essential step towards achievement of this goal is to precisely define the functional roles played by the myelin basic proteins (MBPs) over the course of the myelinogenesis program that is implemented by the oligodendrocyte. For many of the studies outlined below, we will rely on the cDNA transfection systems we have developed and refined over the past two years, that employ specific MBP antisera and confocal immunomicroscopy to map intracellular distributions of the expressed proteins.
Our specific aims are: I.To identify the functional domains within the 14K and 18.5K MBPs that mediate association of these isoforms with the plasma membrane. Using site-directed mutagenesis, we will (1) delete internal segments or (2) truncate the N or C terminus of the 14K and 18.5K MBP cDNAs, and express the mutated constructs in non-glial cells, and in shiverer oligodendrocytes. the intracellular distribution of each mutated polypeptide will be mapped immunocytochemically to determine minimum polypeptide lengths and sequences required for membrane association. In parallel, (3) the mutated polypeptides will be expressed and tested in biochemical and ultrastructural experiments for the capacity to bind to and aggregate oriented vesicles derived from shiverer brain plasma membranes. II.To begin to explore the mechanisms by which the exon II-containing 21.5K and 17K MBPs are translocated from their site of synthesis in the cell cytoplasm across the nuclear pore complex into the nuclear matrix. In non-glial cells, and in shiverer mouse oligodendrocytes transfected with the 21.5K or 17K MBP cDNAs, the expressed proteins are found within the cytoplasm and nucleoplasm. Significantly, in the normal developing mouse brain, oligodendrocytes are readily detected whose nuclei contain high concentrations of MBPs. In many cell types, proteins that are translocated into the nucleus contain elements within their primary amino acid sequences that act as nuclear localization signals (NLSs) which enable selective entry through the nuclear pore complex into the nuclear compartment where the translocated proteins can exert regulatory functions. We have identified a region in the exon II-containing MBP isoforms that bears strong homology to known NLSs. We will test the capacity of this, and other sequences in these MBPs to function as authentic NLSs in transient transfectants. Three criteria that are used to rigorously define NLSs in other systems will be applied: (1) NLS-mediated entry of MBP into the nucleus is likely to be an energy-requiring process, and therefore should be sensitive to disruption by (a) chilling, or (b) ATP depletion; (2) deletion or mutation of MBP NLS sequence(s) should cause cytoplasmic accumulation of the mutated protein; and (3) when the putative MBP NLS sequence is engineered into an unrelated non-nuclear protein, such as bovine serum albumin, the chimeric protein should be translocated into the nucleus.
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