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J. Cell Biol.,
Volume 141, Number 1, April 6, 1998 71-84
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.
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