The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

Abstract

The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca(2+)-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipid membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.

Fig. 1

(A) Amino acid sequence and exon arrangement of the classic 18.5-kDa MBP from the mouse. The exons are delimited by alternating solid or dashed lines. Basic residues are colored blue and acidic residues are colored red. The amino acid numbering omits the N-terminal methionyl residue which is cleaved post-translationally, and follows our standard convention [–17]. (B) ClustalW2 alignment of the conserved regions encoded by exon-II of 21.5-kDa MBP in various species. Here, the residue numbers are primed to distinguish them from the 18.5-kDa numbering in panel (A) for which we have established a convention. Two putative PY-NLS motifs are identified with the pattern of ZX_2–5PB, where “Z” is a basic residue, “X” is any residue, and “B” is a hydrophobic residue. The amino acid residues which were subjected to substitution are highlighted in red. These residues are highly conserved among these species, with the exception of the histidine residue found in the second putative motif in the rabbit genome. The 21-nucleotide 3′-untranslated region (UTR) is the minimal sequence responsible for transport and localization of MBP mRNA within OLGs, including the transfected N19-cells used here [–22]. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Fluorescence intensity trace analysis of nucleocytoplasmic distribution of 21.5-kDa MBP in the N19-OLG cell line. All cells were transfected with 2.0 μg of RFP-tagged plasmid DNA and a nuclear stain, DAPI, seen in blue. Only the merged images are shown. Each trace indicates fluorescence intensity in arbitrary units, versus distance in pixels. The blue line represents DAPI staining, and the red line represents MBP. Each cell is representative of the predominant protein localization phenotype observed in all of the transfected cells observed. Transfection with RFP-tagged 21.5-kDa MBP (panel A) shows little cytosolic presence of 21.5-kDa MBP, indicating its transport to the nucleus, as previously observed [–22]. The same pattern can be seen in cells transfected with RFP-tagged 21.5-kDa MBP constructs containing the single substitutions ΔP67′G, ΔP76′G, and ΔR73′G (panels B, C, and F, respectively). Cells transfected with constructs containing tandem ΔP67′G and ΔP76′G substitutions, or a single ΔK62′G substitution, (panels D and E, respectively), show a slight shift from nuclear to cytoplasmic presence of 21.5-kDa MBP. Scale bar = 40 μm. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Fluorescence micrographs and intensity trace analysis of nucleocytoplasmic distribution of 21.5-kDa MBP in the N19-OLG cell line. All cells were transfected with 2.0 μg of RFP-tagged plasmid DNA and a nuclear stain, DAPI, seen in blue. Each trace indicates fluorescence intensity in arbitrary units, versus distance in pixels. The red fluorescence images are shown in the first column, whereas merged images are shown in the second column. Transfection with RFP-tagged 21.5-kDa MBP with the substitutions (A) ΔK62′E, (B) ΔR73′E, and (C) ΔK62′E and ΔR73′E, shows both nuclear and cytosolic presence of 21.5-kDa MBP, indicating a decrease in nuclear-localization of the protein. Each trace indicates fluorescence intensity in number of pixels. The blue line represents DAPI staining, and the red line represents 21.5-kDa MBP. Each cell is representative of the predominant protein localization phenotype observed in all (100%) of the transfected cells observed. Scale bar = 20 μm. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)