Brain Ac39/physophilin: cloning, coexpression and colocalization with synaptophysin.
Physophilin is an oligomeric protein that binds the synaptic vesicle protein synaptophysin constituting a complex that has been hypothesized to form the exocytotic fusion pore. Microsequencing of several physophilin peptides putatively identified this protein as the Ac39 subunit of the V-ATPase. Ac39 has recently been shown to be present in a synaptosomal complex which, in addition to synaptophysin, includes the bulk of synaptobrevin II, and subunits c and Ac115 of the V0 sector of the V-ATPase. We have cloned physophilin from mouse brain and found a differential 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 occurred in the previous cloning study, and shows that the amino acid sequences of physophilin and Ac39 are completely identical. In situ hybridization in rat brain reveals a largely neuronal distribution of Ac39/physophilin mRNA which spatio-temporally correlates with those of subunit c and synaptophysin. Immunohistochemical analysis shows that Ac39/physophilin is mostly concentrated in the neuropil with a pattern identical to subunit A and very similar to synaptophysin. Double-labelling immunofluorescence shows a complete colocalization of Ac39/physophilin with subunit A and a partial colocalization with synaptophysin in the neuropil. Our findings bring anatomical support for the in vivo occurrence of the synaptophysin-Ac39/physophilin interaction and further suggest a coordinated transcription of V-ATPase and synaptophysin genes. A putative role of Ac39/physophilin in the inactivation of the V-ATPase by disassembly of its V1 sector is also discussed.
Carrión-Vázquez
M
M
Neural Plasticity Department, Instituto Cajal (CSIC), Madrid, Spain. carrion.mariano@mayo.edu
Fernández
A M
AM
Chowen
J
J
Nieto-Sampedro
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D001923:Brain Chemistry / Q000235:genetics*; D002417:Cattle; D003001:Cloning, Molecular; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D013569:Synapses / Q000235:genetics / Q000502:physiology; D016708:Synaptophysin / Q000096:biosynthesis* / Q000235:genetics; D025262:Vacuolar Proton-Translocating ATPases
D012333:RNA, Messenger; D016708:Synaptophysin; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases