BRAIN AC39 PHYSOPHILIN - CLONING, COEXPRESSION AND COLOCALIZATION WITH SYNAPTOPHYSIN/

Physophilin is an oligomeric protein that binds the synaptic vesicle p rotein synaptophysin constituting a complex that has been hypothesized to form the exocytotic fusion pore. Microsequencing of several physop hilin peptides putatively identified this protein as the Ac39 subunit of the V-ATPase. Ac39 has recently been shown to be present in a synap tosomal complex which, in addition to synaptophysin, includes the bulk of synaptobrevin II, and subunits c and Ac115 of the V-0 sector of th e V-ATPase. We have cloned physophilin from mouse brain and found a di fferential region of 12 amino acids when compared with the previously reported sequence of Ac39 from bovine adrenal medulla. RT-PCR cloning from the bovine adrenal medulla demonstrates that sequencing errors oc curred in the previous cloning study, and shows that the amino acid se quences of physophilin and Ac39 are completely identical. In situ hybr idization in rat brain reveals a largely neuronal distribution of Ac39 /physophilin mRNA which spatio-temporally correlates with those of sub unit c and synaptophysin. Immunohistochemical analysis shows that Ac39 /physophilin is mostly concentrated in the neuropil with a pattern ide ntical to subunit A and very similar to synaptophysin. Double-labellin g immunofluorescence shows a complete colocalization of Ac39/physophil in with subunit A and a partial colocalization with synaptophysin in t he neuropil. Our findings bring anatomical support for the in vivo occ urrence of the synaptophysin-Ac39/physophilin interaction and further suggest a coordinated transcription of V-ATPase and synaptophysin gene s. A putative role of Ac39/physophilin in the inactivation of the V-AT Pase by disassembly of its V-1 sector is also discussed.