The synapse-specific phosphoprotein F1-20 is identical to the clathrin assembly protein AP-3.
F1-20 and AP-3 are independently described, synapse-associated, developmentally regulated phosphoproteins with similar apparent molecular masses on SDS-polyacrylamide gel electrophoresis (PAGE). F1-20 was cloned and characterized because of its synapse specificity. AP-3 was purified and studied biochemically because of its function as a clathrin assembly protein. Here we present evidence that establishes the identity of F1-20 and AP-3. Monoclonal antibodies against F1-20 and AP-3 both specifically recognize a single protein from mouse brain with an apparent molecular mass of 190 kDa on SDS-PAGE. These monoclonal antibodies also specifically recognize the cloned F1-20 protein expressed in Escherichia coli. The anti-F1-20 monoclonal antibody (mAb) stains a bovine protein with an apparent molecular mass on SDS-PAGE of 190 kDa that copurifies with brain clathrin-coated vesicles (CCVs) and that can be extracted from the brain CCVs under conditions that extract AP-3. The anti-F1-20 and anti-AP-3 mAbs specifically recognize the same spot on a two-dimensional gel run on a bovine brain clathrin-coated vesicle extract. AP-3 purified from bovine brain CCVs is recognized by both the anti-F1-20 and anti-AP-3 mAbs. Purified preparations of bovine AP-3 and bacterially expressed mouse F1-20 give identical patterns of protease digestion with bromelain and subtilisin. Sequence analyses reveal that F1-20 has an essentially neutral 30-kDa NH2-terminal domain with an amino acid composition typical of a globular structure and an acidic COOH-terminal domain rich in proline, serine, threonine, and alanine. This is consistent with proteolysis experiments that suggested that AP-3 could be divided into a 30-kDa globular uncharged clathrin-binding domain and an acidic, anomalously migrating domain.
Zhou
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S
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
Tannery
N H
NH
Yang
J
J
Puszkin
S
S
Lafer
E M
EM
D033942:Adaptor Proteins, Vesicular Transport; D017398:Alternative Splicing; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D001923:Brain Chemistry; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D010750:Phosphoproteins / Q000096:biosynthesis / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D000596:Amino Acids; D000911:Antibodies, Monoclonal; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; C439023:clathrin assembly protein AP180; D012313:RNA