Fission yeast epsin, Ent1p is required for endocytosis and involved in actin organization.
In this study, we have characterized an essential gene ent1+ encoding fission yeast epsin, which is similar to mammalian and budding yeast endocytic protein epsins. The S. pombe Ent1p contains ENTH (epsin amino-terminal homology) domain at its amino terminus, two copies of a ubiquitin-interacting motif (UIM) immediately carboxyl-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 expression of ent1+ gene, the conditional ent1 gene knockout cells showed a marked defect in internalization of fluorescent dyes, suggesting that Ent1p is essential for endocytosis. Changes in conserved amino acid residues within ENTH domain in ent1 mutant cells revealed temperature-sensitive defect in actin organization and cell morphology. The Ent1p bound PI(4,5)P2 and PI(3,5)P2 immobilized onto nitrocellulose in vitro and also weakly bound PI(3,4)P2, PI(3,4,5)P3, PI4P and PI5P. Surprisingly, the localization of Ent1p-GFP was not affected even in the its3-1 cells, in which the level of PI(4,5)P2 was severely reduced, suggesting that PI(4,5)P2 may not be essential for proper localization of Ent1p at endocytic sites. Our findings indicate that S. pombe Ent1p is an essential component in endocytic process, and involved in actin organization and cell morphogenesis.
Sakamoto
Chika
C
Faculty of Health Sciences, Kobe University School of Medicine, Kobe, Japan.
Kawamoto
Chitose
C
Takeuchi
Kumiko
K
Miyamoto
Ikumi
I
Shuntoh
Hisato
H
D000199:Actins / Q000502:physiology*; D000595:Amino Acid Sequence; D004705:Endocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000166:cytology; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship
D000199:Actins; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins