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J Biol Chem, Vol. 275, Issue 5, 3288-3295, February 4, 2000
From Clathrin-coated pit (CCP) formation occurs as a
result of the targeting and assembly of cytosolic coat proteins, mainly
the plasma membrane clathrin-associated protein complex (AP-2) and clathrin, to the intracellular face of the plasma membrane. In the
present study, the mechanisms by which Eps15, an AP-2-binding protein,
is targeted to CCPs was analyzed by following the intracellular localization of Eps15 mutants fused to the green fluorescent protein. Our previous results indicated that the N-terminal Eps15
homology (EH) domains are required for CCP targeting. We
now show that EH domains are, however, not sufficient for targeting to
CCPs. Similarly, neither the central coiled-coil nor the C-terminal AP-2 binding domains were able to address green fluorescent protein to
CCPs. Thus, targeting of Eps15 to CCPs likely results from the
collaboration between EH domains and another domain of the protein. An
Eps15 mutant lacking the coiled-coil domain localized to CCPs showing
that Eps15 dimerization is not strictly required. In contrast, Eps15
mutants lacking all AP-2 binding sites showed a dramatic decrease in
plasma membrane staining, showing that AP-2 binding sites, together
with EH domains, play an important role in targeting Eps15 into CCPs.
Finally, the effect of the Eps15 mutants on
clathrin-dependent endocytosis was tested by both
immunofluorescence and flow cytometry. The results obtained showed that
inhibition of transferrin uptake was observed only with mutants able to
interfere with CCP assembly.
Mapping of Eps15 Domains Involved in Its Targeting to
Clathrin-coated Pits*
§¶,
,, and INSERM E9925, Faculté Necker-Enfants
Malades, 156 rue de Vaugirard, 75730 Paris, and
§ Unité de Biologie des Interactions Cellulaires,
URA-CNRS 1960, Institut Pasteur, 25 rue de Dr. Roux, 75724 Paris, Cedex
15, France
*
This work was supported by grants from the Association pour
la Recherche contre le Cancer (ARC), from the Fondation Princesse Grace
de Monaco, and from Human Frontier Science Program (grant number
RG404/96).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by Ligue Nationale contre le Cancer.
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