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Proteins interacting with the ascidian vitelline‐coat sperm receptor HrVC70 as revealed by yeast two‐hybrid screening
Author(s) -
Harada Yoshito,
Sawada Hitoshi
Publication year - 2007
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20616
Subject(s) - biology , vitelline membrane , transmembrane protein , sperm , yeast , microbiology and biotechnology , transmembrane domain , two hybrid screening , fusion protein , serine protease , genetics , gene , receptor , protease , biochemistry , embryo , oocyte , recombinant dna , enzyme
Ascidians are hermaphrodites releasing sperm and eggs nearly simultaneously, but many species are self sterile. We have previously reported that HrVC70 consisting of 12 EGF‐like repeats is a major component of the vitelline coat, functioning as a self/nonself‐recognizable sperm receptor during fertilization of the ascidian Halocynthia roretzi. Here, in order to identify the binding partner of HrVC70, we explored HrVC70‐interacting proteins by yeast two‐hybrid screening. HrVC70 is capable of interacting with HrVC70 precursor HrVC120 itself and also with three additional extracellular and/or transmembrane proteins, HrVLP‐1, ‐2, and HrTTSP‐1. Specific interaction of HrVC120, HrVLP‐1, ‐2, and HrTTSP‐1 with HrVC70 was confirmed by exchanging prey and bait, and also by a pulldown assay using the GST‐fusion proteins. HrVLP‐1 and ‐2 are proteins structurally related to HrVC120; both are expressed in the oocytes and may be novel components of the ascidian vitelline coat. HrTTSP‐1 appears to be a member of the serine protease family with type II transmembrane topology. HrTTSP‐1 is expressed in the testis and its gene product contains multiple conserved motifs known to be involved in protein–protein or protein–carbohydrate interactions. Close inspection revealed that the protease domain of HrTTSP‐1 is considerably divergent, in particular around the region of the catalytic center Ser residue. Possible roles of these proteins in ascidian fertilization are also discussed. Mol. Reprod. Dev. 74: 1178–1187, 2007. © 2007 Wiley‐Liss, Inc.