Abstract
A self-association reaction involving the plasma membrane-associated clathrin assembly protein AP-2 has been detected by incubating AP-2 alone under solution conditions that would favor the assembly of complete coat structures if clathrin were present. Self-association was rapid, unaffected by nonionic detergents, readily reversible, and gave rise to sedimentable aggregates. Only the AP subtype AP-2 exhibited self-association: the structurally or functionally related assembly proteins AP-1 and AP-3 and unrelated proteins neither self-associated nor were incorporated into the AP-2 aggregate. AP-2 interactions responsible for self-association were of high affinity, with an apparent Kd of approximately 10(-8)M. By proteolytic dissection, the self-association domain was localized to the core of the molecule containing the intact 50- and 16-kDa polypeptides in association with the truncated 60-66-kDa moieties of the parent alpha/beta polypeptides. Self-association of the intact AP-2 molecule was pH-dependent, exhibiting an apparent pKa approximately 7.4. While it is unlikely that the large AP-2 aggregates formed in solution are themselves biologically relevant structures, the AP-2 interactions involved in their formation have properties consistent with their occurrence in intact cells and thus may be important in cellular functions of the plasma membrane-localized assembly protein.
Highlights
From the Fels Institute for Cancer Research and Molecular Biology and the Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
TheAP subtype AP-2 exhibited selfassociation: the structurally or functionraelllaytedassembly proteinsAP- 1 and AP-3 and unrelated proteins neither self-associatednor were incorporated into the Clathrin coats are peripherally associated with the cytoplasmic surfaces of membranes and can be stripped from isolated coated vesicles under a number of conditions [15,16,17] including 2 M urea, high concentrations of protonated amines
Tures, the AP-2 interactions involved in their formation have properties consistent with their occurrence in intact cells and may be important in cellular functions of the plasma membrane-localized assembly protein
Summary
TheAP subtype AP-2 exhibited selfassociation: the structurally or functionraelllaytedassembly proteinsAP- 1 and AP-3 and unrelated proteins neither self-associatednor were incorporated into the Clathrin coats are peripherally associated with the cytoplasmic surfaces of membranes and can be stripped from isolated coated vesicles under a number of conditions [15,16,17] including 2 M urea, high concentrations of protonated amines The interactionsof the coat proteins with both the membrane the self-association domaiwnas localized to the coorfe and with each other andtherefore give rise to i n ~ v i d u aflree themoleculecontainingtheintact 50- and 16-kDa clathrin triskelions and AP molecules.
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