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.

Figure 1

Degradation of the 70-kDa component (HrVC70) of the H. roretzi vitelline coat by the ubiquitin–proteasome system. (A) HrVC70 was specifically degraded by the ascidian sperm extract. The ¹²⁵I-labeled vitelline coat (VC) was incubated with or without the sperm extracts (2 and 4 μl), followed by SDS/PAGE and autoradiography (Left). Note that the amount of HrVC70 is decreased by addition of the sperm extract in a concentration-dependent manner (Right). A small amount of 45-kDa band was observed in the isolated VC preparation probably because of possible contamination of a small quantity of nonself sperm during the collection process of eggs from H. roretzi, a hermaphroditic animal. (B) HrVC70 was degraded in the presence of 2 mM ATP by the prior addition of ubiquitin (Ub) and ubiquitin-conjugating enzymes [E1, E2, and E3 (E1/2/3)] purified from rabbit reticulocyte lysate followed by the addition of the purified 26S-like proteasome (26S). HrVC70 and its degradation fragments were clearly detected by Western blotting (WB) by using an antibody specific to HrVC70. A 66-kDa faint band is an artifact or a nonspecific band observed in the E1/2/3 preparation. (Ub)n-VC70, high-molecular-weight ubiquitinated HrVC70; Ub-VC70 fragment, ubiquitinated 45-kDa fragment. (C) The same samples as in B were subjected to Western blotting (WB) with the FK2 monoclonal antibody. The 45-kDa fragment was immunoreacted with FK2.

Figure 2

Structure and expression of the HrVC70 precursor protein HrVC120. (A) Deduced amino acid sequence of HrVC120. An N-terminal sequence of HrVC70 determined by Protein Sequencer (brown), a C-terminal Arg residue of HrVC70 (purple), potential O-fucosylation sites (blue), potential N-glycosylation sites (magenta), and a potential furin cleavage site (green) are also indicated. (B) The domain structure of HrVC120. TM domain, transmembrane domain. (C) Northern blotting of VC120. Gn, gonad; Ms, muscle; Bb, branchial basket; In, intestine; Hp, hepatopancreas; Hc, hemocytes.

Figure 3

(A) Similarity of the EGF-like repeats in HrVC70. Consensus sequences in the odd- (CONS-5, 7, 9) and even-numbered EGF-like repeats (CONS-6, 8, 10) in HrVC70 are indicated. Two Lys residues in HrVC70 are shown by blue marks, and cysteine residues are indicated by magenta. (B) Alignment by clustal w of the ZP domain in HrVC120 with ZP domain-containing molecules including human sperm receptor ZP3, mouse ZP3, and Xenopus ZP3 homologue (gp43). (C) Binding of H. roretzi sperm to the HrVC70-immobilized agarose beads. HrVC70 isolated from the vitelline coat with 1 mM HCl or the extract with 50 mM 4-morpholinepropanesulfonic acid (pH 8.0) as a control was coupled to Affi-Gel-10 beads according to the manufacturer's protocol (≈0.5 mg HrVC70/ml beads). The HrVC70-immobilized agarose beads or the control beads in 500-μl suspension were incubated with sperm suspension (100 μl) for 2 h at 13°C. After repeated washing with artificial seawater, the number of sperm bound to one HrVC70-immobilized agarose bead or the control bead was counted in the presence of 4′,6-diamidino-2-phenylindole under a fluorescence microscope with UV excitation.

Figure 4

In vitro ubiquitination of GST-HrVC70 fragment fusion proteins by H. roretzi sperm exudate. The GST-fusion proteins carrying the HrVC70 fragment (GST-VC70_227–270 WT) and its Lys-to-Arg mutant [GST-VC70_227–270 (K234R)] were incubated with the sperm exudate in the presence of 5 mM ATP, ¹²⁵I-ubiquitin, 10 mM MgCl₂, and 1 mM MG115 (proteasome inhibitor). After incubation, the reaction mixtures were subjected to SDS/PAGE, followed by autoradiography.

Figure 5

In vivo ubiquitination of HrVC70 in the vitelline coat of H. roretzi eggs on insemination with sperm. (A) The vitelline coats (VCs) were isolated from the unfertilized and fertilized (5 min after insemination) eggs, and then subjected to SDS/PAGE, followed by Western blotting (WB), by using anti-HrVC70-peptide antibody. (B) The same membrane in A was reprobed with FK2 antibody. (Ub)n-VC70, high-molecular-weight ubiquitinated HrVC70.

Figure 6

Immunocytochemistry of the H. roretzi vitelline coat before (1, 3) and after insemination (2, 4) by using the FK2 antibody as a primary antibody and the FITC-conjugated anti-mouse IgG goat antibody as a secondary antibody. Upper (1, 2) and Lower (3, 4) represent the fluorescence and bright field, respectively, under a fluorescence microscope. Note that the vitelline coat in fertilized eggs showed a strong fluorescence because of ubiquitinated proteins (2). No appreciable fluorescence was observed with a control IgG in either case (data not shown). It is evident that in situ ubiquitination takes place during fertilization. VC, vitelline coat; F, follicle cells; PS, perivitelline space. (Bar = 10 μm.)