Differential sorting of SNAP-25a and SNAP-25b proteins in neuroblastoma cells.
Exocytosis is mediated by high-affinity interactions between different SNARE proteins. The existence of several variants of each SNARE protein suggests that the specificity of fusion may be directed by unique combination of SNARE family members. We examined if two alternatively spliced variants of synaptosomal-associated protein of 25 kD, SNAP-25a and SNAP-25b, possessed distinct cellular distribution if coexpressed within the same neuroblastoma cell. Double-labelling immunofluorescence histochemistry in combination with confocal laser microscopy of individual cell clones revealed a different subcellular localisation pattern for the two SNAP-25 variants. Sucrose density gradient centrifugation of cell homogenates followed by Western blotting showed that the SNAP-25 protein variants associated with intracellular organelles of different density. Taken together, this study shows that two alternatively spliced variants of SNAP-25, differing in only nine amino acids, possess distinct properties at the level of intracellular trafficking, suggesting that the cellular localisation of SNAP-25 protein is regulated at the level of mRNA splicing.
Andersson
J
J
Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden.
Fried
G
G
Lilja
L
L
Meister
B
B
Bark
C
C
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D008565:Membrane Proteins; D051379:Mice; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D012313:RNA / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D014162:Transfection; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C494209:Snap25 protein, mouse; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D012313:RNA