VARIABLE SUBCELLULAR-LOCALIZATION OF A NEURON-SPECIFIC PROTEIN DURINGNTERA-2 DIFFERENTIATION INTO POSTMITOTIC HUMAN NEURONS

The current report describes the molecular characterization of the hum an (the D4S234 locus) and mouse (the m234) homologs of a gene that was isolated during our genomic analysis of the Huntington disease gene r egion. Sequence comparisons of full-length cDNA clones revealed that t he mouse and human homologs encoded evolutionarily conserved 21-kDa pr oteins with greater than 90% amino acid sequence identity. Extensive s equence identity between the D4S234 gene and the rat p1A75 gene (a pre viously identified rat neuron-specific gene) showed that these genes a re interspecies homologs. Furthermore, the D4S234 protein exhibited si gnificant amino acid similarity to a 19-kDa mouse protein that localiz es to the Golgi apparatus of embryonic neurons. However, nonconservati ve sequence differences suggested that these genes are independent mem bers of a multigene family. Northern analyses revealed that rodent D4S 234 expression occurred predominantly in the brain and included all br ain regions. Neuron-specific expression was demonstrated using Norther n analysis of cultured glial cells and quinolinic acid-treated rat bra in samples. Minimal amounts of the rodent D4S234 mRNA were detected pr enatally; however, elevated adult levels were detected within 1 month of birth. Sequence analyses of the human and mouse D4S234 proteins ide ntified an evolutionarily conserved hydrophobic sequence and a consens us nuclear localization signal in both genes. Immunofluorescence micro scopy, using an antipeptide antibody, established that the human D4S23 4 protein preferentially localized to the nucleus of mitotic cultured cells. Since the rat p1A75 protein was previously mapped to the neuron al cytoplasm by in situ hybridization, the subcellular localization of the D4S234 protein was subsequently examined during differentiation o f the NTera 2 (NT2) cell line. Following differentiation into postmito tic NT2-N neurons, the D4S234 protein demonstrated cytoplasmic stainin g and reduced or undetectable nuclear staining in many cells. The vari ation in the intracellular localization of the D4S234 protein in mitot ic and nonmitotic cells suggests that the subcellular localization of this protein is developmentally regulated and provides clues about the biochemical function of this protein.