Two distinct populations of synaptic-like vesicles from rat brain

In nonneuronal cells, several plasma membrane proteins such as exofacial enzymes, receptors, and ion channels recycle between their intracellular compartment(s) and the cell surface via an endosomal pathway. In neurons, however, this pathway has not been extensively characterized. In particular, it remains unclear whether or not it is related to the recycling of small synaptic vesicles, the major pathway of membrane traffic in nerve terminals. To approach this problem, we purified and studied a vesicular fraction from rat brain synaptosomes. Two distinct populations of vesicles with different buoyant densities and sedimentation coefficients were detected in this fraction by sucrose gradient centrifugation and Western blot analysis of the individual proteins. Both populations contain proteins that are markers of synaptic vesicles, namely, SV2, synaptotagmin, synaptophysin, secretory carrier membrane proteins (SCAMPs), synaptobrevin, and rab3a. A striking difference between the two populations is the presence of arginine aminopeptidase activity (a previously suggested marker for the regulated endosomal recycling pathway) exclusively in the lighter less-dense vesicles. The same two vesicular populations were also detected in the preparation of clathrin-coated vesicles isolated from whole rat brain or purified synaptosomes after removal of their clathrin coats by incubation at pH 8.5. We conclude, therefore, that both types of vesicles recycle in synaptosomes via a clathrin-mediated pathway. These data present experimental evidence for biochemical heterogeneity of synaptic-like vesicles in rat brain.