Calbindin_28kDa is specifically associated with extranuclear constituents of the dense particulate fraction

Abstract.

Recent attempts to understand the function of calbindin_28kDa, a widely expressed calcium-binding protein, are confounded by uncertainties over its subcellular location. Using immunoblot analysis of rat brain subregions, we found that the proportion of particulate calbindin_28kDa (24–43% of total) was independent of expression level and location. The association of calbindin_28kDa with particulate structures appeared to be specific, since it persisted when soluble calbindin_28kDa was sequestered by antibodies added before tissue disruption. Moreover, when exogenous calbindin_28kDa was added during homogenisation of brain from calbindin_28kDa-nullmutant mice, only 10% partitioned to the particulate fraction compared with 33% of endogenous calbindin_28kDa in wild-type controls. Confocal microscopy showed that calbindin_28kDa was predominantly extranuclear in all tissues analysed (i.e. various brain regions, isolated neurons, and dental enamel epithelium). Dual-label microscopy of neural dense particulate fractions confirmed the extranuclear location of calbindin_28kDa and also showed that it partly colocalised with synaptosome and microtubule markers. Using sucrose step gradients, calbindin_28kDa was separated from nuclei in parallel with synaptosome and endoplasmic reticulum markers. However, no association with the marker proteins (synaptophysin, ERp29, α/β-tubulin) was detected by calbindin_28kDa-immunoprecipitation analysis. Together these findings provide the first consistent picture that calbindin_28kDa is located predominantly outside of the nucleus, irrespective of tissue type (neuronal vs non-neuronal) and experimental approach (biochemical vs morphological). The evidence of a substantial, strong and specific association with insoluble cellular structures challenges the widely held view of calbindin_28kDa as a mobile calcium buffer, and supports the existence of important alternative roles that involve target proteins.