Proteins interacting with the ascidian vitelline-coat sperm receptor HrVC70 as revealed by yeast two-hybrid screening.
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.
Harada
Yoshito
Y
Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Sugashima, Toba, Japan. yharada@bio.nagoya-u.ac.jp
Sawada
Hitoshi
H
D000595:Amino Acid Sequence; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D004527:Egg Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005306:Fertilization; D008297:Male; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014561:Urochordata / Q000378:metabolism*; D014817:Vitelline Membrane / Q000378:metabolism*
D018076:DNA, Complementary; D004527:Egg Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C035027:egg surface sperm receptor; C035651:vitelline membrane proteins