Cloning and expression of gamma-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus.
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.
Robinson
M S
MS
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, England.
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
D033982:Adaptor Protein Complex gamma Subunits; D011506:Proteins; D004247:DNA