Compartmentalization of clathrin in synaptic terminals

Immunofixation of sodium lauryl sulphate (SDS)-acrylamide gels has been used to study the distribution of the major protein (clathrin) of coated vesicles in various compartments of synaptic terminals. Synaptosomal subcellular fractions were isolated and purified from pig brain homogenates by the procedure of Ueda et al. and lysed in 6 mM Tris-Cl buffer at pH 6.6, 7.8, and 8.1. The synaptosomal particulate and soluble fractions were separated by centrifugation. The synaptic junctional complex (SJC) and postsynaptic density (PSD) fractions were obtained by detergent treatment of the synaptic plasma membrane (SPM). The synaptosomal subcellular fractions and purified coated vesicle (PCV) fractions were subjected to SDS gel electrophoresis (7.5%). The resulting slabs were divided vertically into 4 segments which were stained with Coomassie blue dye, or immunofixed with preimmune and anti-clathrin serum, or affinity labeled with concanavalin A (Con A)-peroxidase. The Comassie blue stained gel indicated the presence of 180,000-molecular weight band in gels of most synaptosomal subcellular fractions. However, immunofixation of an identical gel revealed positive staining of the 180,000-molecular weight protein in PCV, synaptosomal (SF), SPM and synaptoplasmic (SS) fractions only. These findings not only support the contention that a pool of cytosolic-coated vesicle protein is localized at synaptic terminals, they also indicate that clathrin appears highly unlikely to contribute to the structural frameworks of the SJC and PSD of mature synapses.