Two separate domains in the golli myelin basic proteins are responsible for nuclear targeting and process extension in transfected cells

Abstract

The golli products of the myelin basic protein (MBP) gene are expressed in neurons and oligodendrocytes (OLs). In certain neuronal populations, golli proteins undergo translocation between the nucleus and cytoplasm/ processes during development. The proteins consist of two domains, a golli domain of 133 amino acids and an MBP domain of variable length. One objective of this study was to identify the sequences responsible for nuclear targeting. Site-directed mutagenesis and deletion analyses were used to generate a series of golli-green fluorescent protein (GFP) DNA constructs that were transfected into OL and neuronal cell lines to follow localization by confocal microscopy. The results indicated that a 36-residue stretch in the MBP domain is essential for nuclear targeting, and the sequence appears to be a nontraditional localization signal motif. The studies also revealed that overexpression of golli proteins could induce dramatic changes in cell morphology. In OL lines, overexpression of intact golli proteins, or golli peptide alone, caused an increase in the length and number of processes, and the elaboration of membrane sheets. In the neuronal lines, there was a dramatic increase in number and length of extensions. The results, consistent with the timing of golli expression in cells during neural development, suggest that golli proteins may be involved in process formation/extension in OLs and neurons during development. These studies have defined two functional domains in the golli protein. Sequences in the MBP domain target the protein into the nucleus and sequences within the golli domain induce process sheet extension in OLs and neurons.

Region-Specific Myelin Pathology in Mice Lacking the Golli Products of the Myelin Basic Protein Gene

“…After growing for 21 d in vitro, the cells were rinsed briefly in PBS and fixed in 4% buffered paraformaldehyde for 30 min at room temperature. After rinsing in PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature and then processed for immunocytochemistry following the protocol as outlined by Reyes et al (2002). Essentially, fixed cells were incubated in a blocking solution (5% goat se-rum in PBS) followed by an overnight incubation at 4°C with a polyclonal antibody for MBP (1:700).…”

“…They have been found to be expressed during embryonic development in neuronal populations during process extension and migration (Landry et al, 1998). Overexpression of golli induces a striking morphological effect on enhanced process and sheet elaboration in OL cell lines (Reyes and Campagnoni, 2002).…”

Increased Expression of Golli Myelin Basic Proteins Enhances Calcium Influx into Oligodendroglial Cells

“…Golli proteins first appear in many neurons when they are extending processes for migration, establishment of connections and, in the case of oligodendrocytes (OLs), before myelination (Landry et al, , 1997(Landry et al, , 1998Pribyl et al, 1996). In vitro transfection studies have shown that overexpression of golli proteins in OL cell lines can induce these cells to elaborate extensive processes and membrane sheets, adopting a morphology similar to mature OLs in culture (Reyes and Campagnoni, 2002).…”

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

“…It has also been suggested that the phosphorylation status of several potential protein kinase C (PKC) phosphorylation sites within the N-terminal region of MBP may modulate their subcellular localization in OLGs [20,22,[31][32][33]45], another phenomenon worthy of further exploration.…”

“…It has been shown that Golli proteins, which arise from transcription start site 1 of the same gene complex as MBP, but are expressed earlier in development, have a non-traditional nuclearlocalization signal (NLS) contained within amino acids 1-36 of the classic MBP portion (Fig. 1A) [33]. However, this non-traditional NLS encoded within exon-I of classic MBP does not cause 18.5-kDa MBP to be localized in the nucleus, and presumably is also not responsible for the localization of 21.5-kDa MBP in the nucleus.…”