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PMID: 8654387 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

In vitro binding of clathrin adaptors to sorting signals correlates with endocytosis and basolateral sorting.

The EMBO journal ·Vol. 15 ·No. 11 ·1996-06-03 ·Pages 2893-9

Heilker R, Manning-Krieg U, Zuber JF, Spiess M

Abstract

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.

MeSH Terms
Adaptor Protein Complex alpha Subunits Adaptor Proteins, Vesicular Transport Amino Acid Sequence Animals Cattle Cell Polarity Clathrin/metabolism Endocytosis Hemagglutinins, Viral/metabolism Kinetics Membrane Proteins/metabolism Molecular Sequence Data Peptides/metabolism
Chemicals
Adaptor Protein Complex alpha Subunits Adaptor Proteins, Vesicular Transport Clathrin Hemagglutinins, Viral Membrane Proteins Peptides
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Heilker R
Biozentrum, University of Basel, Switzerland.
Manning-Krieg U
Zuber J F
Spiess M
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43 references, click to expand
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1996-06-03
Pages
2893-9
Language
English
Region
England
NLM ID
8208664
PMCID
PMC450227
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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