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.fertilization ͉ vitelline coat ͉ ubiquitin F ertilization is a key event in sexual reproduction, creating a new individual with novel genomic information. In animal reproduction, species-specific binding of sperm to the proteinaceous egg coat, called the vitelline coat in marine invertebrates or the zona pellucida in mammals, is particularly important for successful fertilization. Because the egg coat is a potential barrier to sperm-egg fusion, sperm must use a lytic agent (lysin) to penetrate it (1, 2). In mammals, it has long been believed that the sperm acrosomal trypsin-like protease acrosin is a zona lysin, which digests zona pellucida proteins to enable sperm to penetrate through the zona pellucida (3). However, recent studies by using acrosin-knockout mice revealed that acrosin is not essential for fertilization or sperm penetration through the zona pellucida (4). Rather, it is currently thought that acrosin is involved in the dispersal of acrosomal proteins during acrosome reaction (5) and that a sperm protease(s) other than acrosin is the actual zona-lysin in mammalian fertilization (6).Ascidians (Urochordata) occupy a phylogenetic position between invertebrates and segmented vertebrates. They are hermaphrodites that usually release sperm and eggs simultaneously during the spawning season. Several ascidians, including Ciona intestinalis (7) and Halocynthia roretzi (8), strictly prohibit selffertilization. Because self-nonself recognition in fertilization is accomplished by interaction between the sperm and vitelline coat (7,8), the sperm-lysin system seems to be triggered after the sperm recognizes the vitelline coat as nonself...

Ascidians are hermaphrodites releasing sperm and eggs simultaneously, but many species are self-sterile because of a self͞nonself-recognition system in sperm-egg interaction. Here, we show that a 70-kDa vitelline coat protein, HrVC70, consisting of 12 epidermal growth factor-like repeats, plays a key role in self͞nonself recognition during ascidian fertilization. We discovered that the amount of HrVC70 of the self-sterile mature oocytes is markedly higher than that of the self-fertile immature oocytes and that the selfsterile mature oocytes become self-fertile by acid treatment, which is able to release the HrVC70 from isolated vitelline coats. In addition, fertilization is strongly inhibited by the pretreatment of sperm with HrVC70 from a different individual, but not from the same individual, and the number of nonself sperm bound to HrVC70-agarose was significantly higher than that of self-sperm. A sequence analysis of HrVC70 disclosed that several amino acid residues in a restricted region are substituted at an individual level, with no identical sequences among the 10 individuals tested. Furthermore, genomic DNA analysis revealed that the epidermal growth factor-like domains correspond to the exons, and each intron is highly conserved among even-and odd-numbered introns, suggesting that multiple gene duplications or amplification of this region might have taken place during evolution. It was also found that diversity in cDNA sequences is derived from genomic DNA polymorphism probably elicited by crossing over and specific nucleotide substitutions. These results indicate that HrVC70 is a candidate allogeneic recognition molecule in the gamete interaction of the ascidian Halocynthia roretzi.

The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.

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The molecular chaperone function of HSP90 is activated under heat-stress conditions. In the present study, we investigated the role of the interactions in the heat-induced activation of HSP90 molecular chaperone. The preceding paper demonstrated two domain-domain interactions of HtpG, an Escherichia coli homologue of mammalian HSP90, i.e. an intra-molecular interaction between the N-terminal and middle domains and an intermolecular one between the middle and C-terminal domains. A bacterial two-hybrid system revealed that the two interactions also existed in human HSP90a. Partners of the interaction between the N-terminal and middle domains of human HSP90a could, but those between the middle and C-terminal domains could not, be replaced by the domains of HtpG. Thus, the interface between the N-terminal and middle domains is essentially unvaried from bacterial to human members of the HSP90-family proteins. The citrate synthase-binding activity of HtpG at an elevated temperature was solely localized in the N-terminal domain, but HSP90a possessed two sites in the N-terminal and other domains. The citrate-synthase-binding activity of the N-terminal domain was suppressed by the association of the middle domain. The complex between the N-terminal and middle domains is labile at elevated temperatures, but the other is stable even at 70 8C. Taken together, we propose the liberation of the N-terminal client-binding domain from the middle suppressor domain is involved in the temperature-dependent activation mechanism of HSP90 molecular chaperone.Keywords: 90-kDa heat-shock protein (HSP90); HtpG; molecular chaperone; peptide binding; heat-induced activation.The molecular chaperone activity of the 90-kDa heat shock protein (HSP90) was first characterized in the terms of the function and regulation of steroid hormone receptors [1][2][3]. The cast of the target substrates has subsequently grown to include various types of proteins involved in signal transduction [4 -6] and even retrovirus reverse transcriptase [7] (reviewed in [8,9]). HSP90 acts in cooperation with other chaperones and cochaperones, such as immunophilins, CDC37/p50, HSP70, p23, Hip, Hop/p60 and PA28 [10 -12] (reviewed in [8,9] On the other hand, HSP90 is composed of three domains at the primary structure level [23,25]. We designated them domain A, Met1-Arg400; domain B, Glu401-Lys615; and domain C, Asp621 -Asp732 [23]. We previously proposed a model of an HSP90 dimer in which domain B of one subunit is associated with domain C of another subunit in an antiparallel fashion [18]. This model indicates that the terminal globule of an HSP90 dimer is composed of one domain A and that the interior globule composed of one domain B of one subunit and one domain C of another subunit.In the preceding paper, we demonstrated that the domain structure of HtpG:, i.e. domain A, Met1-Arg336; domain B, Glu337 -Lys552; and domain C, Leu553-Ser624, is essentially identical to that of human HSP90a. Hence, it is reasonable to postulate that the basic mechanism of the members of ...

Organs consist of the parenchyma and stroma, the latter of which coordinates the generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulation of complex organ configurations from pluripotent stem cells (PSCs) have remained challenging. By elucidating the in vivo molecular features of the renal stromal lineage at a single-cell resolution level, we herein establish an in vitro induction protocol for stromal progenitors (SPs) from mouse PSCs. When the induced SPs are assembled with two differentially induced parenchymal progenitors (nephron progenitors and ureteric buds), the completely PSC-derived organoids reproduce the complex kidney structure, with multiple types of stromal cells distributed along differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived lineage-specific stroma into parenchymal organoids will pave the way toward recapitulation of the organotypic architecture and functions.

SUMMARY1. To clarify the mechanism which regulates the time course of twitch tension when /1-and muscarinic receptors are stimulated, intracellular Ca2+ transients, Ca21 sensitivity of the contractile element and the cross-bridge cycling rate (CCR) were measured in ferret ventricular muscles. 4. In order to measure CCR, the perturbation analysis method was applied to steady-state tension of tetanic contraction. The CCR was not altered even when the tetanic tension level was decreased to 50 % by decreasing [Ca2+]0. Iso (0-1 taM) slightly decreased the tetanic tension level and increased the CCR from 2 73 to 3 25 Hz. The effect of Iso was observed when the Iso-decreased tension was recovered by an increase in [Ca2+]i. The addition of ACh (1 /tM) recovered the CCR which was increased by Iso, to the control level. Atropine (10 yM) blocked the effect of ACh, and carbachol (1 /tM) restored the CCR increased by Iso to the control level.5. The time course of Ca21 transients, Ca2+ sensitivity and CCR were antagonistically regulated by fl-and muscarinic receptor stimulation, but the time course of tension did not parallel the changes in these parameters. Therefore, these results suggest that the time course of tension, particularly the relaxation time, is not determined by the time course of Ca2+ transients, Ca2+ sensitivity and the CCR, and that other factors might be involved in the regulation of the time course of tension when /3-and muscarinic receptors are stimulated.S98 26

) and C-terminal one-third of the middle region were sufficient for the interactions with the N-and C-terminal regions, respectively. Yeast HSC82 that carried point mutations in the middle region causing deficient binding to the N-terminal region could not support the growth of HSP82-depleted cells at an elevated temperature. Taken together, our data show that the N-terminal and middle regions of the HSP90 family protein are structurally divided into two respective subregions. Moreover, the interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 in yeast.The 90-kDa heat shock protein (HSP90) 1 has been demonstrated to be an important molecule, chaperoning a variety of cellular proteins, such as steroid receptors (1-3), protein kinases involved in signal transduction (4 -6), and even retrovirus reverse transcriptase (7) and endothelial nitric-oxide synthase (8) (for reviews, see Refs. 9 and 10). HSP90 occupies a central part of the chaperone network, the "foldsome," and functions in cooperation with other chaperones and co-chaperones, such as immunophilins, CDC37/p50, HSP70, p23, Hip, Hop/p60, and PA28 (11-13) (for reviews, see Refs. 9 and 10).This assembly process of the HSP90-substrate protein complex requires ATP (14, 15), which induces a conformational change in HSP90 (16 -18). Recently, it was demonstrated that HSP90 is capable of linking substrates for degradation by the ubiquitin-proteasome pathway by cooperating with the E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP) (19 -21). Thus, HSP90 may play a central role in deciding the fate of proteins, refolding or degradation.HSP90 family proteins are composed of three regions at the primary structure level (22,23). In the present study using human HSP90␣, we denote the N-terminal region, Region B and Region C mediate dimerization of the HSP90 family proteins; Region B of one subunit is associated with Region C of another subunit in an antiparallel fashion (22). Electron microscopy showed that an HSP90 dimer consists of four linearly arranged globules (18), and the N-and C-terminal immunogenic sites (23) were localized in the terminal and interior globules, respectively (28).To accomplish the molecular function of HSP90, each region may have additional roles that should be unveiled. For instance, although the ATP binding site has been localized toward the amino terminus of HSP90, ATP binding as well as elevated temperature bring about a profound conformational change that is not restricted to the ATP-binding domain (16 -18). When the concentration of HSP90 was lower than 1 M, both ATP binding and elevated temperature induced an equivalent conformational change, converting HSP90 from a linear dimer into an O-ring-shaped structure (18). On the other hand, when the concentration of HSP90 was sufficiently high, HSP90 self-oligomerized instead of formed O-ring-shaped molecules, probably through essentially identical interactions (29). Alteration of the regional interaction may be closely related to these ...