Assembly and targeting of adaptin chimeras in transfected cells

Abstract

Adaptors are the components of clathrincoated pits and vesicles that attach the clathrin to the membrane. There are two types of adaptors in the cell: one associated with the plasma membrane and one associated with the TGN. Both adaptors are heterotetramers consisting of two adaptins (α and β for the plasma membrane; γ and β′ for the TGN), plus two smaller proteins. The COOH-terminal domains of the adaptins form appendages that resemble ears, connected by flexible hinges. Unlike the other adaptor components, the COOH termini of the α- and γ-adaptins show no homology with each other, suggesting that they might provide the signal that directs the adaptors to the appropriate membrane. To test this possibility, the COOH-terminal ears were switched between α- and γ-adaptins and were also deleted. All of the constructs contained the bovine γ-adaptin hinge, enabling them to be detected with a species-specific antibody against this region when transfected into rat fibroblasts. Immunoprecipitation indicated that the engineered adaptins were still fully capable of assembling into adaptor complexes. Immunofluorescence revealed that in spite of their modified ears, the constructs were still able to be recruited onto the appropriate membrane; however, the ear-minus constructs gave increased cytoplasmic staining, and replacing the γ-adaptin ear with the α-adaptin ear caused a small amount of colocalization with endogenous α-adaptin in some cells. Thus, the major targeting determinant appears to reside in the adaptor "head," while the ears may stabilize the association of adaptors with the membrane.