Dysbindin and pallidin protein levels are higher in the cerebral cortex and hippocampus (Hippo.) of C57BL/6J mice at postnatal day (P)1, and in cerebellum (Cerebell.) from P7 mice, than in the corresponding brain regions of young adult (P45) mice. “Whole-tissue” detergent extracts were analyzed by immunoblotting using antibodies against the indicated proteins. The immunoreactivity signals derived from densitometric analysis were corrected for background and expressed as a ratio to the sum of background-corrected signals obtained in the same immunoblot for one sample each of the three brain regions of P45 mice. Each bar represents mean value ± s.e.m. of 4–5 independent brain samples. One-way ANOVA followed by Bonferroni’s multiple comparison tests: ** P < 0.01, *** P < 0.001, ns, not significant.

SNARE complex assembly impairs BLOC-1 binding. (a) Schematic representation of recombinant proteins used to assemble a Qa/Qb/Qc/R SNARE domain complex consisting of full-length SNAP-25b, the cytoplasmic region of VAMP2, and the SNARE domain region of various syntaxins. The GST and His₆-S (His) tags are depicted out of scale. (b) SDS-PAGE analysis of recombinant proteins used for the binding assay, including SNARE complexes containing SNAP-25b, the cytoplasmic region of VAMP2 and fragments of syntaxins 1, 4 or 13 (numbered arrows). The amounts of loaded proteins correspond to those used for the binding assay. Notice the presence of slow-migrating species (asterisk) in aliquots of the complexes that were processed for SDS-PAGE without boiling to reveal the existence of SDS-resistant SNARE complexes. (c) The purified SNARE complexes, unassembled SNAP-25b corresponding to 25–100% of the amount incorporated into the complexes, and additional control proteins were immobilized on affinity beads and incubated with bovine brain cytosol. Proteins in the washed beads, as well as aliquots of the cytosol, were analyzed by immunoblotting using a mAb against the pallidin subunit of BLOC-1. Notice that the amounts of BLOC-1 bound to SNARE complexes were significantly less than that bound to unassembled SNAP-25b.

Developmental regulation of dysbindin protein level in mouse cerebral cortex. (a) Immunoblotting analysis of “whole-tissue” detergent extracts (20 μg total protein) prepared from cerebral cortices dissected out of C57BL/6J mice at embryonic day (E)14 and E18 and postnatal day (P)1 through P45. (b) Quantitative analyses of the relative expression levels of selected proteins in cortex from mice at different developmental ages. Each value derived from densitometric analysis was corrected for background and divided by the sum of values obtained in the same immunoblot image for one sample each of P1, P7, P14 and P21. For the sake of clarity, data are represented as means ± s.e.m. (2–4 independent samples per age), even when all data points were analyzed together by linear regression to assess departure from a theoretical line with slope equal to zero (18 degrees of freedom; dysbindin, P < 0.001; β-actin, not significant).

Interaction of brain BLOC-1 with recombinant SNARE proteins. (a, c, d, f) Purified GST-fusion proteins were immobilized on glutathione-Sepharose beads and incubated with bovine brain cytosol. Following extensive washing, bound proteins were eluted from beads and analyzed by immunoblotting using a mAb against the pallidin subunit of BLOC-1. Aliquots of the cytosolic extract, corresponding to 0.1–3% of the total amount incubated with each GST-fusion protein, were analyzed in parallel. (a) Both SNAP-25a and -25b interacted robustly with BLOC-1 under conditions in which SNAP-23 and -29, as well as the cytoplasmic regions (cyt) of the Qb SNAREs Vti1a and Vti1b, failed to bind detectable amounts of the complex. (b) Schematic representation of the SNAP-25b protein, which contains the SNARE domains Qb and Qc separated by a linker region. (c) Both the Qb domain and a portion of the linker, but not the Qc domain, were necessary for robust binding of SNAP-25b to BLOC-1. No BLOC-1-binding activity was observed for recombinant proteins bearing the cytoplasmic regions of SNARE proteins of the VAMP family. (e) Schematic representation of the syntaxin 13 protein, which contains an auto-inhibitory Habc domain, a Qa SNARE domain and a transmembrane (TM) region. (d, f) The SN fragment of syntaxin 13, but not the entire cytoplasmic fragment containing the Habc domain, interacted selectively with BLOC-1. SDS-PAGE analyses of all recombinant proteins used in these experiments are shown in Supplementary Figure S3.

Absence of BLOC-1 results in neurite outgrowth defects in hippocampal neurons. (a) Representative images of primary hippocampal neurons cultured from wild-type (C57BL/6J) and BLOC-1-deficient (pallid) mice in Tii medium for 1 or 3 days. Cells were fixed in 4% (w/v) paraformaldehyde and stained with antibodies against GAP43 (red). DAPI staining was used to label the cell nuclei (blue). Corresponding phase images are shown. Astrocytes are denoted with “A”. Bars, 50 μm. (b–d) Primary hippocampal neurons were grown in Tii medium (circles) or DMEM medium plus serum (triangles). The number of neurites (b), length of the longest neurite (c) and sum of the lengths of all neurites (d) per neuron were analyzed using a blinded approach. Each pair of symbols connected by a line represents the median values of 15–64 randomly selected images analyzed for wild-type (empty symbols) and pallid mutant (filled symbols) cells in each independent preparation. See Supplementary Table S1 and Figure S6 for further details. Statistical significance was assessed using an ANOVA model as described under Materials and methods. ** P < 0.01, *** P < 0.001, ns, not significant.

Evidence for the existence of brain dysbindin as a stable component of BLOC-1. (a, b) The steady-state protein levels of dysbindin are significantly decreased in brain from BLOC-1-deficient, pallid (pa) mutant mice. “Whole-tissue” detergent extracts were prepared from cerebral cortex, hippocampus and cerebellum dissected from mice at postnatal day (P)45 (a), and from cerebral cortex and hippocampus dissected from P1 mice (b), and then analyzed by immunoblotting using antibodies against the indicated proteins. Besides the major ~50-kDa dysbindin form, minor species of ~42 and ~38 kDa were detected. (c) Reductions in dysbindin protein levels at steady state were also noted in primary hippocampal neurons from pallid mice cultured in Tii medium for 1 or 3 days. (d) Mouse brains were homogenized in the absence of detergents, and the homogenates subjected to ultracentrifugation to obtain a soluble protein fraction and a membrane pellet. Notice that the bulk of dysbindin was recovered from the soluble fraction. (e, f) Soluble protein fractions prepared from P7 brain (e) and adult brain (f) were further fractionated by size-exclusion chromatography, and the resulting fractions were analyzed by immunoblotting using antibodies to dysbindin, pallidin, and a monomeric dysbindin-related protein named CK1BP. The exclusion volume (Vo) and elution positions of proteins with known Stokes’ radii (in Angstroms) are indicated on the top. The elution profile of dysbindin, consisting of a peak corresponding to large Stokes’ radii (95±5 Å, arrowheads), was in close match to that of pallidin (e) and drastically different from that of CK1BP (f).