title: Cloning and expression of gamma-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus.
abstract: Adaptins are the major components of adaptors, the protein complexes that link clathrin to transmembrane proteins (e.g., receptors) in coated pits and vesicles. The plasma membrane adaptor contains an alpha-adaptin subunit and a beta-adaptin subunit, while the Golgi adaptor contains a gamma-adaptin subunit and a beta'-adaptin subunit. A partial cDNA clone encoding gamma-adaptin was isolated from a bovine brain expression library by screening with antibodies, and was used to obtain a cDNA clone from a mouse brain library containing the full coding sequence. The identity of the clones was confirmed by protein sequencing. The deduced amino acid sequence of gamma-adaptin was found to be homologous to that of alpha-adaptin, with several stretches of identical amino acids or conservative substitutions in the first approximately 70 kD, and 25% identity overall. Weaker homology was seen between gamma- and beta-adaptins. Like both alpha- and beta-adaptins, gamma-adaptin has a proline and glycine-rich hinge region, dividing it into NH2- and COOH-terminal domains. A chimeric gamma-adaptin was constructed from the mouse and bovine cDNAs and transfected into Rat 1 fibroblasts. Immunofluorescence microscopy was carried out using an mAb which recognizes an epitope present on the chimera but not found on the rodent protein. The construct was found to have a distribution typical of endogenous gamma-adaptin. Using this transfection system, it should now be possible to exchange domains between alpha- and gamma-adaptins, to try to find out how adaptors are targeted to the appropriate membrane compartment of the cell, and how they recruit the appropriate receptors into the coated vesicle.
authors: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, England.
authors: M S
authors: MS
authors: Robinson
mesh_terms: D033982:Adaptor Protein Complex gamma Subunits; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D006056:Golgi Apparatus / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
chemical_list: D033982:Adaptor Protein Complex gamma Subunits; D011506:Proteins; D004247:DNA