Variable subcellular localization of a neuron-specific protein during NTera 2 differentiation into post-mitotic human neurons

The current report describes the molecular characterization of the human (the D4S234 locus) and mouse (the m234) homologs of a gene that was isolated during our genomic analysis of the Huntington disease gene region. Sequence comparisons of full-length cDNA clones revealed that the mouse and human homologs encoded evolutionarily conserved 21-kDa proteins with greater than 90% amino acid sequence identity. Extensive sequence identity between the D4S234 gene and the rat p1A75 gene (a previously identified rat neuron-specific gene) showed that these genes are interspecies homologs. Furthermore, the D4S234 protein exhibited significant amino acid similarity to a 19-kDa mouse protein that localizes to the Golgi apparatus of embryonic neurons. However, nonconservative sequence differences suggested that these genes are independent members of a multigene family. Northern analyses revealed that rodent D4S234 expression occurred predominantly in the brain and included all brain regions. Neuron-specific expression was demonstrated using Northern analysis of cultured glial cells and quinolinic acid-treated rat brain samples. Minimal amounts of the rodent D4S234 mRNA were detected prenatally; however, elevated adult levels were detected within 1 month of birth. Sequence analyses of the human and mouse D4S234 proteins identified an evolutionarily conserved hydrophobic sequence and a consensus nuclear localization signal in both genes. Immunofluorescence microscopy, using an antipeptide antibody, established that the human D4S234 protein preferentially localized to the nucleus of mitotic cultured cells. Since the rat p1A75 protein was previously mapped to the neuronal cytoplasm by in situ hybridization, the subcellular localization of the D4S234 protein was subsequently examined during differentiation of the NTera 2 (NT2) cell line. Following differentiation into postmitotic NT2-N neurons, the D4S234 protein demonstrated cytoplasmic staining and reduced or undetectable nuclear staining in many cells. The variation in the intracellular localization of the D4S234 protein in mitotic and nonmitotic cells suggests that the subcellular localization of this protein is developmentally regulated and provides clues about the biochemical function of this protein.