title: Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor.
abstract: The ubiquitin-proteasome system is essential for intracellular protein degradation, but an extracellular role of this system has not been known until now. We have previously reported that the proteasome is secreted into the surrounding seawater from sperm of the ascidian (Urochordata) Halocynthia roretzi on sperm activation, and that the sperm proteasome plays a key role in fertilization. Here, we show that a 70-kDa component (HrVC70) of the vitelline coat is the physiological substrate for the ubiquitin-proteasome system during fertilization of H. roretzi. A cDNA clone encoding the HrVC70 precursor (HrVC120) was isolated, and a homology search revealed that HrVC120 contains 13 epidermal growth factor-like repeats and a mammalian zona pellucida glycoprotein-homologous domain. HrVC70 functions as a sperm receptor. We demonstrate that HrVC70 is ubiquitinated both in vitro and in vivo. The immunocytochemical localization of multiubiquitin chains in the vitelline coat and the inhibitory effect of monoclonal antibodies against the multiubiquitin chains on fertilization strongly support the role of the ubiquitin-proteasome system in ascidian fertilization. Taken together, these results indicate that the ubiquitin-proteasome system is responsible for extracellular degradation of the sperm receptor HrVC70 and, consequently, for sperm penetration of the vitelline coat during fertilization.
authors.affiliation: Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812 Japan. hswd@pharm.hokudai.ac.jp
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sawada
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Sakai
authors.forename: Yukichi
authors.initials: Y
authors.lastname: Abe
authors.forename: Etsuko
authors.initials: E
authors.lastname: Tanaka
authors.forename: Youko
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Junko
authors.initials: J
authors.lastname: Fujino
authors.forename: Eri
authors.initials: E
authors.lastname: Kodama
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takizawa
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Yokosawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005110:Extracellular Space / Q000502:physiology; D005260:Female; D005306:Fertilization; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013084:Sperm-Ovum Interactions / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*; D014561:Urochordata / Q000502:physiology*; D014817:Vitelline Membrane / Q000502:physiology
chemical_list: D009097:Multienzyme Complexes; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; C035027:egg surface sperm receptor; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex