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Vol. 16, Issue 2, 717-730, February 2005

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Interaction of Sla2p's ANTH Domain with PtdIns(4,5)P₂ Is Important for Actin-dependent Endocytic Internalization

Yidi Sun ^*, Marko Kaksonen ^*, David T. Madden , Randy Schekman , and David G. Drubin ^* 

^* Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202; Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720-3202

Submitted August 25, 2004; Accepted November 23, 2004
Monitoring Editor: Anthony Bretscher

A variety of studies have implicated the lipid PtdIns(4,5)P₂ in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P₂-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P₂ binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P₂ specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P₂ binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P₂ levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P₂ binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P₂ interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P₂ binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P₂ plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Corresponding author. E-mail address: drubin{at}socrates.berkeley.edu.

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