Cloning of Drosophila beta-adaptin and its localization on expression in mammalian cells.
A Drosophila cDNA (BAD1) encoding a structural and assembly-competent homologue of the mammalian coated pit beta-adaptins (beta and beta') has been cloned and sequenced. In its amino-terminal region (residues 1-575), the BAD1 sequence appears intermediate between that of the mammalian beta-adaptin and a predicted sequence, from cDNA 105a, which appears to code for a version of beta'-adaptin. To test its functional characteristics, a 'myc'-tagged version of BAD1 was expressed in Cos cells. The BAD1 protein was detected most clearly in plasma membrane coated pits, where it colocalized with alpha-adaptin, although other coated pits were noted which apparently did not contain alpha-adaptin. However, these are probably gamma-adaptin containing pits, as BAD1 was also found colocalized with gamma-adaptin in Golgi coated pits in which, typically, alpha-adaptin is absent. Immunoprecipitation experiments confirmed that the BAD1 protein was present in both types of adaptor complex, unlike beta-adaptin which complexes with alpha-adaptin and beta'-adaptin which partners gamma-adaptin exclusively. In spite of this, BAD1 expression does not appear to mix alpha-adaptin and gamma-adaptin distribution amongst all the coated pits: thus the location of these adaptor complexes in mammalian cells does not depend on the differences between beta subunits but rather on membrane-specific interactions of other adaptor polypeptides. The differential interaction of beta with alpha-adaptin and beta' with gamma-adaptin in mammalian cells is likely to depend on the few non-conservative differences between their respective sequences and BAD1. Four of these (one with respect to beta and three versus 105a) are clustered in a particular region (residues 155 to 305), which may therefore represent a domain that influences the choice of partner adaptin.
Camidge
D R
DR
Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK.
Pearse
B M
BM
D033961:Adaptor Protein Complex 1; D033966:Adaptor Protein Complex beta Subunits; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D004247:DNA; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins; D005455:Fluorescent Antibody Technique; D016259:Genes, myc; D006801:Humans; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D033961:Adaptor Protein Complex 1; D033966:Adaptor Protein Complex beta Subunits; C439125:Bap protein, Drosophila; D029721:Drosophila Proteins; D011506:Proteins; D004247:DNA