Purification of the synaptic vesicle-binding protein physophilin. Identification as 39-kDa subunit of the vacuolar H(+)-ATPase.
Physophilin is a 36-kDa polypeptide originally identified in synaptic plasma membrane fractions, which binds to synaptic vesicles and has been implicated in vesicle docking and/or exocytosis during neurotransmitter release. Here we report on the purification, amino acid sequence analysis, and subcellular localization of physophilin. Physophilin was enriched from detergent extracts of crude synaptic plasma membranes by a combination of cation exchange and lentil-lectin chromatography. Sequence analysis of peptides generated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that physophilin is identical to the 39-kDa subunit (Ac39) of the vacuolar H(+)-ATPase. This was confirmed further by Western blot analysis with an Ac39-specific antiserum and by vesicle binding assays with recombinant Ac39 protein. Subcellular fractionation showed that Ac39 is enriched in synaptic vesicles, with lesser amounts being present in synaptic plasma membrane fractions. These results argue against a docking role of physophilin/Ac39 in synaptic vesicle exocytosis.
Siebert
A
A
Abteilung Neurochemie, Max-Planck-Institut für Hirnforschung, Frankfurt/Main, Germany.
Lottspeich
F
F
Nelson
N
N
Betz
H
H
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009479:Neuropeptides / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D014176:Protein Biosynthesis; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013570:Synaptic Membranes / Q000201:enzymology; D013572:Synaptic Vesicles / Q000201:enzymology*
D046911:Macromolecular Substances; D009479:Neuropeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D006180:Proton-Translocating ATPases