Fission Yeast Epsin, Ent1p is Required for Endocytosis and Involved in Actin Organization

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In this study, we have characterized an essential gene ent1+ encoding fission yeastepsin, which is similar to mammalian and budding yeast endocytic protein epsins. TheS. pombe Ent1p contains ENTH (epsin amino-terminal homology) domain at its aminoterminus, two copies of a ubiquitin-interacting motif (UIM) immediatelycarboxyl-terminal to the ENTH domain, three NPF motifs in the carboxyl-terminal half,and the clathrin-binding motif at the carboxyl terminal. When repressed the expressionof ent1+ gene, the conditional ent1 gene knockout cells showed a marked defect ininternalization of fluorescent dyes, suggesting that Ent1p is essential for endocytosis.Changes in conserved amino acid residues within ENTH domain in ent1 mutant cellsrevealed temperature-sensitive defect in actin organization and cell morphology. TheEnt1p bound PI(4,5)P2 and PI(3,5)P2 immobilized onto nitrocellulose in vitro and alsoweakly bound PI(3,4)P2, PI(3,4,5)P3, PI4P and PI5P. Surprisingly, the localization ofEnt1p-GFP was not affected even in the its3-1 cells, in which the level of PI(4,5)P2 wasseverely reduced, suggesting that PI(4,5)P2 may not be essential for proper localizationof Ent1p at endocytic sites. Our findings indicate that S. pombe Ent1p is an essentialcomponent in endocytic process, and involved in actin organization and cellmorphogenesis.
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