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First published online 17 February 2009
doi: 10.1242/jcs.036343
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Research Article |
Department of Physiology, Hebrew University, Hadassah Medical School, Jerusalem, 91120, Israel
* Author for correspondence (e-mail: millet_t{at}cc.huji.ac.il)
Accepted 5 November 2008
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Summary |
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Key words: C. elegans, Proteasome, CUL-3
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Introduction |
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). This process is assisted by a number of ER-resident chaperones, including nonspecific chaperones such as BIP and calnexin, and the nAChR-specific chaperone RIC-3 (Blount and Merlie, 1991; Chang et al., 1997; Halevi et al., 2002). RIC-3 homologs increase the efficiency of the maturation process, increasing both steady-state levels of receptor subunits and surface expression of mature receptors (Halevi et al., 2003; Cheng et al., 2005; Castillo et al., 2005; Cohen Ben-Ami et al., 2005). Effects of RIC-3 homologs are not restricted to quantitative effects, because the human RIC-3 homolog enables maturation of 
7 nicotinic receptors in cells that will otherwise express no functional 7 receptors (Williams et al., 2005), and RIC-3 was also shown to affect properties of the C. elegans DEG-3/DES-2 receptor (Cohen Ben-Ami et al., 2005). Thus RIC-3 family members are key players in the maturation of nAChRs in both vertebrates and invertebrates.
Using a two-hybrid screen in yeast, we identified BATH-42 as a protein that interacts with the RIC-3 C-terminus. BATH-42, which contains BTB/POZ (broad-complex, Tramtrack and bric-a-brac/Pox virus and zinc finger) and MATH (meprin-associated Traf homology) domains, was previously identified to interact with CUL-3 (Xu et al., 2003). CUL-3 functions as a scaffold within a cullin-ring ligase (CRL) complex, whose function is to recruit E2-ubiquitin-conjugating enzymes to specific substrates. Substrate specificity of CRLs requires a substrate receptor (Petroski and Deshaies, 2005). Specifically, for CUL-3, the substrate receptors are BTB-domain proteins. These proteins interact with CUL-3 via their BTB domain and recruit the substrate via another protein-interaction domain (Xu et al., 2003; Petroski and Deshaies, 2005). Similarly to BATH-42, MEL-26 contains BTB and MATH domains, and targets the microtubule-severing protein MEI-1/katanin for CUL-3-dependent degradation, thus enabling meiosis-to-mitosis transition (Pintard et al., 2003).
BATH-42 might therefore also target specific proteins, such as RIC-3, for degradation by the ubiquitin proteasome system. To evaluate this hypothesis, we examined the in vivo implications of interactions between RIC-3 and BATH-42 by analyzing the effects of BATH-42 loss of function or overexpression. This analysis supports our hypothesis, and suggests that interactions between BATH-42 and RIC-3 enable regulation of the levels of RIC-3. Moreover, we show that this mechanism is active under physiological conditions. In addition, we show that excess RIC-3 is deleterious for nAChR function and distribution, motivating the need for such regulation.
Work by others has shown regulation of receptor subunits by the proteasome (reviewed by Yi and Ehlers, 2005). Our work highlights the need to regulate not only the receptors themselves, but also the chaperones required for their assembly, and specifically identifies a mechanism that protects cells from excess RIC-3. Moreover, this suggests that the proteasome indirectly regulates synaptic transmission mediated by nAChRs via regulation of RIC-3.
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Results |
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), which are known to function in protein-protein interactions (Stefancsik et al., 1998). To identify proteins that interact with the RIC-3 C-terminus, we used it as bait in a yeast two-hybrid screen. This screen identified BATH-42 (C50C3.8) as a RIC-3 C-terminus interactor (Fig. 1A). To verify this interaction, we used GST pull-down of in vitro transcribed and translated BATH-42. This analysis validates the two-hybrid results, showing a strong interaction between BATH-42 and the RIC-3 C-terminus when fused to GST (Fig. 1B). Interaction between GST:RIC-3 and BATH-42 is specific, because a much weaker interaction was seen using GST alone or GST fused to the unrelated ODZ protein (Levine et al., 1994) (Fig. 1B). The conserved coiled-coil sequences are likely to be responsible for the observed interactions, because GST fusions and two-hybrid baits containing only the first coiled-coil and short flanking sequences are sufficient for this interaction (results not shown).
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Expression of BATH-42 and RIC-3 overlap
In yeast cells and in vitro, BATH-42 interacts with the RIC-3 coiled-coil domain. For this interaction to occur in C. elegans, the two proteins should be expressed within the same cells. To examine whether BATH-42 expression overlaps with RIC-3 we fused the intergenic sequences upstream of BATH-42 to GFP. Analysis of this bath-42p:GFP reporter shows restricted expression in specific muscles, namely the pharyngeal and the vulval muscles (Fig. 2A,C). Occasionally expression was also seen in a few neurons in the head and body regions. In addition, bath-42p:GFP expression was seen in non-excitable cells, namely the posterior part of the intestine and the seam cells (Fig. 2B). Vulval muscles, pharyngeal muscles, and most neurons are known to express RIC-3 and to require RIC-3 for maturation of nAChRs (Halevi et al., 2002). Specifically, within the pharyngeal muscle, RIC-3 is required for maturation of the EAT-2 nAChR and therefore for pharyngeal pumping, and within the vulva muscles, RIC-3 is required for maturation of the levamisole-sensitive muscle nAChR. Thus an interaction between BATH-42 and RIC-3 within these cells has the potential to affect nAChR-mediated signaling.
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bath-42 loss of function enhances RIC-3 levels and reduces nAChR function in vulval muscles
To examine the functional implications of BATH-42–RIC-3 interaction we studied a bath-42 loss of function [bath-42(lf)] allele made available by the National Bioresource Project, Japan (www.shigen.nig.ac.jp/c.elegans/index.jsp). This mutant was outwardly wild type, with a normal growth rate, the normal number of eggs (Fig. 3B) and a normal rate of pharyngeal contractions [2.9±2.6% of bath-42(lf) were pumping defective relative to 3.3±2.6% of N2 (n=35, N=5 each)]. Thus BATH-42 is not essential for function of the pharyngeal or vulval muscles under normal growth conditions.
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), is decreased (Fig. 3B). This effect is due to a difference in the response to levamisole, because no significant difference in egg laying is seen on food or in M9 buffer without levamisole (Fig. 3B). To show that this second effect is not a result of background mutations in the bath-42(lf) strain, we also examined the effects of bath-42 dsRNA feeding on the egg-laying response to levamisole. Bath-42 knockdown in wild-type N2 animals resulted in a reduction of the egg-laying response to levamisole 60±10% relative to N2 fed with vector dsRNA (n=33-36, N=3, P<0.01). This reduction is similar to the phenotype of bath-42(lf) mutants. Thus, we conclude that reduced BATH-42 expression leads to reduced sensitivity of the egg-laying response to levamisole. Levamisole sensitivity serves as an assay for function and expression levels of the levamisole-sensitive receptors (Waggoner et al., 2000), suggesting that reduced bath-42 expression, as seen in the bath-42(lf) mutant or following treatment with bath-42 dsRNA, leads to a reduced level or function of the vulva muscle receptor. Our results show that bath-42(lf) leads to an increase in RIC-3 levels while reducing expression or function of the levamisole-sensitive nAChR in vulva muscles. The first result is consistent with a role for BATH-42 in targeting RIC-3 to degradation, and shows that in the absence of BATH-42, steady-state levels of RIC-3 increase. However, the second result appears inconsistent with this hypothesis, unless increased RIC-3 expression is detrimental for its function. To examine this possibility, we looked at the effects of overexpressing RIC-3 on the egg-laying response to levamisole. This analysis showed that increased RIC-3 expression, achieved by expressing RIC-3 from different copy numbers of a RIC-3:GFP transgene, was associated with a decrease in the number of eggs laid in response to levamisole (Fig. 4). Thus, decreased degradation of RIC-3 in bath-42(lf) mutants leading to an increase in RIC-3 levels can explain the decreased egg-laying response to levamisole. If indeed bath-42(lf) egg-laying defects are a result of RIC-3 overexpression, we expect that reducing RIC-3 expression will suppress this defect. To examine this prediction, we preformed levamisole-dependent egg-laying assays on bath-42(lf) homozygous animals that are heterozygous for a ric-3(lf) mutation. Results of this assay show that levamisole-dependent egg-laying is increased in these mutants relative to bath-42(lf) mutants carrying two wild-type copies of ric-3 (Fig. 3B). These results support our hypothesis that the effects of bath-42(lf) on levamisole sensitivity are a result of its effects on RIC-3 expression.
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Overexpression of RIC-3 influences nAChR distribution and function
Next, we examined the distribution of overexpressed RIC-3. In vulva muscles, overexpressed RIC-3 accumulated in puncta, which are likely to be aggregates of RIC-3 (Fig. 4B). These RIC-3 aggregates might sequester nAChR subunits away from the assembly process. To examine this suggestion, we looked at effects of RIC-3 overexpression on localization of the DEG-3 nAChR subunit in PVD cell bodies (Yassin et al., 2001). This analysis shows that overexpression of RIC-3 affects the distribution of DEG-3, leading to a less-homogeneous distribution (Fig. 5A,B). Moreover, DEG-3 accumulations overlap with RIC-3 accumulations, supporting the idea that excess RIC-3 sequesters nAChR subunits away from the assembly process. Overexpression of RIC-3 reduced the number of DEG-3(u662)-dependent degenerations, as assayed by counting the number of swollen (degenerating) neurons, i.e. degenerations that are caused by constitutive activity of this mutant nAChR (Fig. 5C) (Treinin and Chalfie, 1995). This effect is consistent with our suggestion that RIC-3 overexpression interferes with RIC-3 function and thus with nAChR distribution and function.
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). Heat-shock induction of BATH-42 expression in adults led to decreased growth (fewer progeny and reduced rate of development). This effect was associated with a large decrease in pharyngeal pumping (Fig. 6), a phenotype similar to that seen in ric-3(lf) mutants (Halevi et al., 2002). Heat-shock of wild-type animals not expressing hsp-16-2p:BATH-42 did not significantly reduce growth rate or pharyngeal pumping (30±4.6% were defective in pumping following heat shock, compared with to 19±2.6 % without heat shock; n=43-46, N=6), showing that this effect required BATH-42 overexpression.
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). Thus we can only examine whether the effects of overexpressing BATH-42 require the conserved coiled-coil domain in pharyngeal muscles but not in vulva muscles. Analysis presented in Fig. 6 shows that animals expressing the RIC-3 TM transgene are resistant to the effects of BATH-42 overexpression. To show that this resistance was not due to the presence of RIC-3 on a multi-copy extrachromosomal array, we also examined the effects of BATH-42 overexpression on another RIC-3 derivative, RIC-3 
N, differing from RIC-3 TM in having an additional 65 amino acids encoding the conserved coiled-coil domain. This RIC-3 derivative rescues the ric-3(lf)-dependent defect as well as wild-type RIC-3 (Biala et al., 2008). Animals expressing this RIC-3 N derivative were sensitive to the effects of BATH-42 overexpression (Fig. 6). Thus we conclude that interaction of BATH-42 with the RIC-3 C-terminus, probably with the conserved coiled-coil domain, is required for effects of BATH-42 overexpression on pharyngeal pumping.
BATH-42 was previously shown to interact with CUL-3, and is thus likely to function as a substrate specific adaptor, targeting specific proteins for degradation in a CUL-3-dependent manner (Xu et al., 2003). Effects of overexpression of BATH-42 on pharyngeal pumping are similar to the effects of ric-3 loss of function. Thus we suggest that BATH-42 targets RIC-3 for degradation in a CUL-3-dependent manner. To examine this, we investigated whether the effects of BATH-42 overexpression are suppressed by depletion of CUL-3. For this purpose, we examined the effects of BATH-42 overexpression in animals fed with cul-3 dsRNA. CUL-3 was required for the effects of BATH-42 overexpression on pharyngeal pumping, because cul-3 dsRNA treatment overnight before heat shock suppressed the effects of BATH-42 overexpression (Fig. 6). Control experiments using dsRNA treatment of vector sequences showed no suppression of the effects of BATH-42 overexpression. Thus, the effects of BATH-42 overexpression require CUL-3. These results, together with our previous work, suggest that the effects of BATH-42 overexpression are likely to be a result of CUL-3-dependent ubiquitylation and degradation of RIC-3.
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Discussion |
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). BTB proteins are postulated to function as substrate-specific receptors, enabling recognition of specific substrates by CUL-3, thereby directing specific proteins to CUL-3-dependent ubiquitylation (Petroski and Deshaies, 2005). Here we show that BATH-42 interacts with RIC-3, an effector of nAChR maturation. Effects of BATH-42 loss of function or overexpression are consistent with a role of BATH-42 in targeting RIC-3 for degradation in a CUL-3-dependent manner. We suggest that under physiological conditions, BATH-42 activity maintains optimal levels of RIC-3 by targeting excess RIC-3 for degradation. Excess RIC-3 interferes with function of nAChRs, and thus, loss of BATH-42 function also interferes with nAChR function.
Members of the RIC-3 family have a key role in nAChR maturation in vertebrates and invertebrates. RIC-3 and its homologs were shown to affect maturation of many different nAChRs. Effects of RIC-3 family members on nAChRs include changes in surface expression, steady-state levels, receptor properties and the formation of functional receptors (Halevi et al., 2003; Williams et al., 2005; Cheng et al., 2005; Castillo et al., 2005; Cohen Ben-Ami et al., 2005). Thus, the regulation of RIC-3 levels is likely to have major effects on nAChR-mediated synaptic signaling.
Moreover, results presented here suggest that overexpression of RIC-3 leads to formation of RIC-3 aggregates that colocalize with nAChR subunits, and are therefore likely to reduce the amount of receptor subunits available for formation of functional receptors. Thus, tight regulation of RIC-3 levels is important. We note that RIC-3 is not the only chaperone whose quantity is regulated by the proteasome. The quantity of UNC-45, a chaperone for muscle myosin, is also regulated by the proteasome (Hoppe et al., 2004). Overexpression of UNC-45, similarly to overexpression of RIC-3, is detrimental for its function. Specifically, overexpression of UNC-45 interferes with myosin assembly (Hoppe et al., 2004). The detrimental effects of overexpression of RIC-3 or UNC-45 suggest that excess chaperone levels sequester interacting proteins away from the assembly process. Indeed, overexpression of functional RIC-3:GFP, in addition to its effects on nAChR function in vulval muscle, and the PVD neurons, also affects nAChR function in body-wall muscles (Y.B., unpublished results). Thus, the maintenance of optimal RIC-3 levels by the proteasome is important for normal cholinergic signaling.
Regulation of synaptic protein turnover by ubiquitylation enables regulation of synaptic plasticity. In post-synaptic membranes ubiquitylation was shown to regulate turnover of glutamate receptors (reviewed by Yi and Ehlers, 2005). Specifically, BTB- and Kelch-domain-containing proteins were shown to affect the levels of glutamate receptors in C. elegans neurons and in mammalian cells (Schaefer and Rongo, 2006; Salinas et al., 2006). In addition to effects of the ubiquitin proteasome pathway on mature receptors within the synapse, this pathway was also shown to affect maturation of receptors. Specifically, proteasome inhibition increased the quantity of nAChRs, suggesting that a balance between degradation and assembly regulates the level of mature nAChRs (Christianson and Green, 2004). RIC-3 family members also affect the level of mature nAChRs (Cheng et al., 2005; Castillo et al., 2005; Cohen Ben-Ami et al., 2005), suggesting that an interaction with RIC-3 protects nAChR subunits from degradation. Therefore, it is possible to regulate the quantity of mature nAChRs both directly, through ubiquitylation and degradation of nAChR subunits and indirectly, through ubiquitylation and degradation of RIC-3. Our results showing increased RIC-3 levels in BATH-42 loss-of-function mutants suggest that RIC-3 levels in vivo are controlled by the proteasome. Such regulation, in addition to protecting cells from the detrimental effects of excess RIC-3, provides the means for regulating the synaptic activity mediated by many different nAChRs.
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Materials and Methods |
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)], was cloned into pAS-1 and served as bait following transformation into strain y1024 (MATa, gla4, trp1-901, ura3-52, ade2-101, URA3::GAL1-lacZ, LYS2::GAL1-HIS3, his3-200 leu-2-3,112, cyhr (kindly provided by Yona Kassir, Technion, Haifa, Israel). The bait-containing cells were then transformed with a C. elegans cDNA 
ACT-RB2 library (kindly provided by Robert Barstead, OMRF, University of Oklahoma, Oklahoma City, OK) and plated on –His, –Leu, –Trp with 20 mM 3-amino-1,2,4-triazole. Colonies growing on these plates contain a bait plasmid and a prey plasmid enabling HIS3 expression. To further examine these colonies they were transferred to nitrocellulose for LacZ assays as described below. Colonies producing blue color in this assay, an indication of LacZ (β-galactosidase) expression, were further studied. Only plasmids that on their own did not enable LacZ expression but did enable LacZ expression upon isolation and retransformation to a bait-containing strain were further analyzed. For LacZ assays shown in Fig. 1, strains were grown on YEPD plates overnight, overlaid with nitrocellulose filters, and then crushed by placing the filter in liquid nitrogen; nitrocellulose filters were then placed on 3MM filter paper saturated with Z-buffer with X-gal, and allowed to develop overnight.
Nematode growth and analysis
Wild-type (N2 Bristol) and all other strains were grown on NGM plates seeded with OP50 at 20°C unless stated otherwise (Wood, 1988). Induction of dsRNA expression was done in LB + ampicillin using 4 mM IPTG for 3 hours. The bacteria (HT115DE3) expressing cul-3, bath-42, or vector dsRNA were then spun down, resuspended using M9 buffer (0.02 M KH2PO4, 0.042 M Na2HPO4, 0.085 M NaCl and 1 mM MgSO4) + 5 mM IPTG, and seeded on NGM plates. For dsRNA feeding experiments, L4 animals were transferred to plates seeded with dsRNA expressing bacteria and grown overnight at 20°C before egg-laying assays or heat-shock and pumping assays. Heat shock was done for 0.5 hours at 32°C followed by 2 hours recovery at 20°C. Pumping rate was observed under high magnification and animals whose pumping rate was lower than 70 pumps per 30 seconds were considered pumping defective (normal pumping rate is 
110 pumps per 30 seconds). Egg-laying experiments were conducted on adults picked the night before to fresh plates and then assayed on fresh NGM plates or in multiwell plates in the presence of M9 or in M9 + 50 µM levamisole, 1 animal per plate or well for 2 hours (Waggoner et al., 2000). bath-42(lf) homozygous that are heterozygous for ric-3(md1181) were obtained by crossing bath-42(lf) males with bath-42(lf) ric-3(md1181) double mutant hermaphrodites, progeny of this cross not showing the ric-3(md1181) phenotypes (coiling movement and other ric-3 loss-of-function phenotypes are recessive) were picked for levamisole assays as described above. For neuronal degeneration assays, the high copy number (20 ng/µl) RIC-3:GFP functional transgene was crossed into a deg-3(u662) background. Degenerations were observed in early L1 larva, 1-2 hours after hatching, using Nomarski optics (Treinin and Chalfie, 1995). As controls, we used larva from the plates that did not carry the transgene, as seen by the absence of the GFP marker. In all experiments n is number of animals examined, and N is the number of independent experiments. Results are given as average ± s.e.m.
Molecular biology
To generate RIC-3:GST fusions, a BamHI site was inserted upstream of the first, conserved, coiled-coil domain following PCR amplification from yk719-F3 (Halevi et al., 2002). Either a BamHI-EcoRI fragment (entire C-terminus) or a BamHI-SalI fragment (first, conserved, coiled-coil domain) was cloned into pGEX-3X. A 1.3 kb EcoRI-BglII fragment of BATH-42 from the two-hybrid isolated clone was cloned into pET-17b for in vitro transcription and translation. Purification of the GST fusions, in vitro expression of labeled proteins, and GST pull-down experiments were done as described (Jimenez et al., 1997). Ponceau staining was performed on gels to confirm that all GST fusions were expressed similarly. BATH-42p:GFP was generated following PCR amplification of a 1.1 kb fragment from cosmid T06F1, which was then cloned into the BamHI and SalI sites of pPD95.75 (Fire et al., 1990). This fragment covers the intergenic region between BATH-42 and C50C3.6 lying upstream to BATH-42. For dsRNA studies, the 1.3kb EcoRI-BglII fragment of BATH-42 from the two-hybrid isolated clone was cloned into the pPD129.36 plasmid, enabling dsRNA expression in bacteria; controls contained vector alone (Fire et al., 1998). For overexpression studies, the coding region of BATH-42 was amplified (and inserted into plasmid pPD49.78 downstream of the hsp16-2 promoter (Jones et al., 1986; Fire et al., 1990). RIC-3 overexpression was achieved by injecting 5 ng/µl or 20 ng/µl of a functional RIC-3:GFP transgene into ric-3(md1181) animals [GFP was inserted in-frame into the SalI site in a ric-3 rescuing clone, this clone rescues ric-3(lf) function (Halevi et al., 2002)]. RIC-3 TM and RIC-3 N transgenics contain the RIC-3 TM or the RIC-3 minimal sequence as previously described (Cohen Ben-Ami et al., 2005) upstream and in-frame of the GFP-coding sequence and between the noncoding regulatory regions of the ric-3 rescuing clone (Halevi et al., 2002). These RIC-3 transgenes were crossed into a strain also containing the hsp-16-2p:BATH-42 transgene and the ric-3(md1181) mutation for RIC-3 TM.
Immunohistochemistry
The C-terminus of RIC-3 fused to GST (see above) was purified as described above, and used to immunize rabbits. Immunohistochemistry was done after picric acid fixation as previously described (Yassin et al., 2001), using 1:1000 dilution of the anti-RIC-3 antibody and 1:250 dilution of Alexa Fluor 488 anti-rabbit antibodies (Invitrogen 48619A). For double staining, we used 1:150 dilution of anti-DEG-3 (Yassin et al., 2001) and anti-GFP (Roche Applied Sciences; 1:50,000). Before staining, RIC-3 antibodies were incubated overnight with ric-3(md1181) animals to remove non-specific antibodies. Images were recorded using a CCD camera (Hamamatsu ORCA ER) and a SimplePCI image-acquisition and analysis program (Compix). Intensity of staining is average intensity per pixel for the vulval muscles normalized to the average intensity per pixel in an adjacent region within the same animal containing body wall muscles and ventral cord neurons. Coefficient of variation is the s.d. divided by the mean intensity of staining within the PVD.
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