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© The Rockefeller University Press, 0021-9525/1998//71 $5.00
The Journal of Cell Biology, Volume 141, Number 1, , 1998 71-84

Regular Articles

Pan1p, Yeast eps15, Functions as a Multivalent Adaptor That Coordinates Protein–Protein Interactions Essential for Endocytosis



Beverly Wendland and Scott D. Emr

Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, University of California at San Diego, School of Medicine, La Jolla, California 92093-0668

A genetic screen for factors required for endocytosis in the budding yeast Saccharomyces cerevisiae previously identified PAN1. Pan1p is a homologue of the mammalian protein eps15, which has been implicated in endocytosis by virtue of its association with the plasma membrane clathrin adaptor complex AP-2. Pan1p contains two eps15 homology (EH) domains, a protein–protein interaction motif also present in other proteins that function in membrane trafficking. To address the role of Pan1p and EH domains in endocytosis, a yeast two-hybrid screen was performed using the EH domain–containing region of Pan1p. This screen identified yAP180A, one of two yeast homologues of a class of clathrin assembly proteins (AP180) that exhibit in vitro clathrin cage assembly activity. In vitro binding studies using GST fusion proteins and yeast extracts defined distinct binding sites on yAP180A for Pan1p and clathrin. yAP180 proteins and Pan1p, like actin, localize to peripheral patches along the plasma membrane. Mammalian synaptojanin, a phosphatidylinositol polyphosphate-5-phosphatase, also has been implicated in endocytosis recently, and three synaptojanin-like genes have been identified in yeast. We observed genetic interactions between the yeast SJL1 gene and PAN1, which suggest a role for phosphoinositide metabolites in Pan1p function. Together with other studies, these findings suggest that Pan1p coordinates regulatory interactions between proteins required for both endocytosis and actin-cytoskeleton organization; these proteins include the yAP180 proteins, clathrin, the ubiquitin–protein ligase Rsp5p, End3p, and synaptojanin. We suggest that Pan1p (and by extension eps15) serves as a multivalent adaptor around which dynamic interactions between structural and regulatory components of the endocytic pathway converge.

Abbreviations used in this paper: CALM, clathrin assembly lymphoid myeloid leukemia gene; chc, clathrin heavy chain; EH, eps15 homology; GFP, green fluorescent protein; IP, inositol polyphosphate; ORF, open reading frame; PI, polyphosphoinositide; YPD, yeast extract-peptone-dextrose.

Address all correspondence to Beverly Wendland or Scott D. Emr, Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, University of California at San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0668. Tel.: (619) 534-7673. Fax: (619) 534-6414. E-mail: bwendland{at}ucsd.edu; semr{at}ucsd.edu


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