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54
Syp1 regulation of actin polymerization and cargo
recognition in Clathrin Mediated Endocytosis
Amanda Reider 1 , Yorke Zhang 1 , Beverly Wendland 1 .
Johns Hopkins University 1
Abstract:
Endocytosis is a dynamic process that is critical for maintaining membrane composition,
regulating transmembrane cargo turnover and nutrient uptake, and controlling signaling
networks. The muniscin family of adaptors is conserved from yeast to humans, with domains
that contribute to cargo selection, membrane tubulation/constriction and actin polymerization;
these activities are coordinated to form an endocytic vesicle. We are currently defining the
mechanisms by which each domain functions individually, and cooperatively, to mediate
endocytosis. Syp1 is one of the earliest proteins recruited to a nascent endocytic site;
interestingly, while Syp1 lacks a classical ‘clathrin box’ motif, its localization to the membrane is
partially dependent upon clathrin. We have found that the C-terminal μ-homology domain that
binds cargo and Ede1 also binds clathrin. We are exploring the role for clathrin in recruiting or
stabilizing Syp1 at the membrane, and possible cooperativity with cargo-recognition. We
identified the transmembrane protein Mid2 as a cargo selected by Syp1, and are now defining
endocytic sorting signals in the Mid2 cytoplasmic tail. Using phage display and recombinant
protein binding, we are searching for other Syp1 cargos as well as the signal for Syp1 cargo
recognition. These approaches will also better define the mode of interaction between Syp1 and
its partner Ede1 (yeast Eps15), and reveal if the interactions of Syp1 with cargo and Ede1 are
independent or competitive. We are also examining the role of the membrane-binding
proline-rich domain of Syp1 in regulating Las17 (yeast WASp) activity and Syp1 membrane
tubulation. We have made a proline-rich domain mutant that ameliorates binding to
membranes and are testing it for effects on actin polymerization and endocytic internalization.
These studies will reveal insights into the temporal coordination of the endocytic process.