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MBC in Press, published online ahead of print December 1, 2004
Mol. Biol. Cell 10.1091/mbc.E04-08-0740

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Submitted on August 25, 2004
Accepted on November 23, 2004

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,{dagger} Randy Schekman,{dagger} and David G. Drubin*{ddagger}

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

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, sla2 mutants 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, multi-color 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.

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






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