IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF THE HPC-1 ANTIGEN IN RAT CEREBELLUM

HPC-1 antigen is a neuron-specific 34 kDa protein, identical to p35A ( syntaxin), and is thought to play important roles in docking or fusion of synaptic vesicles to presynaptic active zones. In the present stud y we analyze the distribution of HPC-1 antigen in rat cerebellum by a cryoimmunogold technique using an antibody against the fusion protein of beta-galactosidase and the HPC-1 antigen. HPC-1 antigen was detecte d at high density on the plasma membranes and synaptic vesicles of pre synaptic boutons which formed synapses with dendrites of Purkinje cell s, and on the plasma membranes of parallel fibres in the cerebellar mo lecular layer. In the granule cell layer, gold particles were also det ected on the endoplasmic reticulum, nuclear membranes and the plasma m embranes of granule cells. Presynaptic membranes and synaptic vesicles in glomeruli were also labelled by gold particles. To determine the t opology of HPC-I antigen on the membranes, the synaptosome fraction pr epared from rat cerebellum was embedded in agarose, and processed for the pre-embedding protein A-gold technique. Intact synaptosomes were n ot labelled by gold particles. However, when fixed in hypotonic fixati ve to rupture plasma membranes, or when ruptured after fixation in nor motonic fixative, the cytoplasmic surfaces of presynaptic membranes an d synaptic vesicles were labelled by gold particles. These results sug gest that most of the epitopes of HPC-1 antigen are located on the cyt oplasmic surface of plasma membranes and synaptic vesicle membranes.