The ENTH and C-terminal domains of Dictyostelium epsin cooperate to regulate the dynamic interaction with clathrin-coated pits.
Epsin contains a phospholipid-binding ENTH domain coupled to C-terminal domain motifs that bind coated pit proteins. We examined how these domains interact to influence epsin function and localization in Dictyostelium. Although not required for global clathrin function, epsin was essential for constructing oval spores during development. Within the epsin protein, we found that features important for essential function were distinct from features targeting epsin to clathrin-coated pits. On its own, the phospholipid-binding ENTH domain could rescue the epsin-null phenotype. Although necessary and sufficient for function, the isolated ENTH domain was not targeted within clathrin-coated pits. The C-terminal domain containing the coated-pit motif was also insufficient, highlighting a requirement for both domains for targeting to coated pits. Replacement of the ENTH domain by an alternative membrane-binding domain resulted in epsin that sequestered clathrin and AP2 and ablated clathrin function, supporting a modulatory role for the ENTH domain. Within the ENTH domain, residues important for PtdIns(4,5)P2 binding were essential for both epsin localization and function, whereas residue T107 was essential for function but not coated pit localization. Our results support a model where the ENTH domain coordinates with the clathrin-binding C-terminal domain to allow a dynamic interaction of epsin with coated pits.
Brady
Rebecca J
RJ
Department of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Wen
Yujia
Y
O'Halloran
Theresa J
TJ
D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000235:genetics / Q000378:metabolism*; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D033761:Spores, Protozoan / Q000166:cytology / Q000235:genetics / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D015800:Protozoan Proteins; C114154:epsin