In vitro binding of clathrin adaptors to sorting signals correlates with endocytosis and basolateral sorting.
To analyze the interaction of sorting signals with clathrin-associated adaptor complexes, we developed an in vitro assay based on surface plasmon resonance analysis. This method monitors the binding of purified adaptors to immobilized oligopeptides in real time and determines binding kinetics and affinities. A peptide corresponding to the cytoplasmic domain of wild-type influenza hemagglutinin, an apical membrane protein that is not endocytosed, did not significantly bind adaptor complexes. However, peptide sequences containing a tyrosine residue that has previously been shown to induce endocytosis and basolateral sorting were specifically recognized by adaptor complexes. The in vitro rates of adaptor association with these peptides correlated with the internalization rates of the corresponding hemagglutinin variants in vivo. Binding was observed both for purified AP-2 adaptors of the plasma membrane and for AP-1 adaptors of the Golgi, with similar apparent equilibrium dissociation constants in the range 10(-7)-10(-6) M. Adaptor binding was also demonstrated for a sequence containing a C-terminal di-leucine sequence, the second major motif of endocytosis/basolateral sorting signals. These results confirm the concept that interaction of cytoplasmic signals with plasma membrane adaptors determines the endocytosis rate of membrane proteins, and suggest the model that clathrin-coated vesicles of the trans-Golgi network are involved in basolateral sorting.
Heilker
R
R
Biozentrum, University of Basel, Switzerland.
Manning-Krieg
U
U
Zuber
J F
JF
Spiess
M
M
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D016764:Cell Polarity; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D006389:Hemagglutinins, Viral / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D006389:Hemagglutinins, Viral; D008565:Membrane Proteins; D010455:Peptides