Prodomain-dependent tissue targeting of an ADAMTS protease controls cell migration in Caenorhabditis elegans
2007; Springer Nature; Volume: 26; Issue: 11 Linguagem: Inglês
10.1038/sj.emboj.7601718
ISSN1460-2075
AutoresShinji Ihara, Kiyoji Nishiwaki,
Tópico(s)Insect Resistance and Genetics
ResumoArticle10 May 2007free access Prodomain-dependent tissue targeting of an ADAMTS protease controls cell migration in Caenorhabditis elegans Shinji Ihara Shinji Ihara RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan Search for more papers by this author Kiyoji Nishiwaki Corresponding Author Kiyoji Nishiwaki RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan Search for more papers by this author Shinji Ihara Shinji Ihara RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan Search for more papers by this author Kiyoji Nishiwaki Corresponding Author Kiyoji Nishiwaki RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan Search for more papers by this author Author Information Shinji Ihara1 and Kiyoji Nishiwaki 1 1RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan *Corresponding author. RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. Tel.: +81 78 306 3262; Fax: +81 78 306 3261; E-mail: [email protected] The EMBO Journal (2007)26:2607-2620https://doi.org/10.1038/sj.emboj.7601718 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted proteins play important roles in animal development and pathogenesis. However, the lack of in vivo models has hampered elucidation of the mechanisms by which these enzymes are recruited to specific target tissues and the timing of their activation during development. Using transgenic worms and primary cell cultures, here we show that MIG-17, an ADAMTS family protein required for gonadal leader cell migration in Caenorhabditis elegans, is recruited to the gonadal basement membrane in a prodomain-dependent manner. The activation of MIG-17 to control leader cell migration requires prodomain removal, which is suggested to occur autocatalytically in vitro. Although the prodomains of ADAMTS proteases have been implicated in maintaining enzymatic latency, polypeptide folding and secretion, our findings demonstrate that the prodomain has an unexpected function in tissue-specific targeting of MIG-17; this prodomain targeting function may be shared by other ADAMTSs including those in vertebrates. Introduction ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family proteins are a group of zinc-dependent metalloproteases (MPs) that mainly degrade extracellular matrix (ECM) components such as proteoglycans and collagens (Colige et al, 1997; Tortorella et al, 1999, 2000; Kuno et al, 2000; Matthews et al, 2000; Fernandes et al, 2001; Sandy et al, 2001; Somerville et al, 2003; Wang et al, 2003). ADAMTSs have common structural features: a signal peptide, a prodomain, an MP domain, a disintegrin (DI) domain, a variable number of thrombospondin type I (TS) motifs and other ancillary domains near the C-terminus (Porter et al, 2005). The prodomain is required for maintaining enzymatic latency in ADAMTS-4 (Tortorella et al, 2005) and probably for polypeptide folding and secretion (Porter et al, 2005). The function of the DI domains is not known. Because ADAMTSs are secreted, they must be brought to their target sites of action during development. In Caenorhabditis elegans, two ADAMTS family proteins, GON-1 and MIG-17, act in gonad development (Blelloch and Kimble, 1999; Nishiwaki et al, 2000). During larval development of C. elegans hermaphrodites, the anterior and posterior gonad arms elongate along the body wall and make two turns as they extend, finally forming the symmetrical U-shaped gonad arms. The migration of gonad arms is directed by two specialized gonadal leader cells, the distal tip cells (DTCs) (Kimble and White, 1981) (Figure 1A). GON-1 and MIG-17 are proposed to be required for proper remodeling of the gonadal basement membrane, which supports active morphogenesis of the gonad (Blelloch et al, 1999; Nishiwaki et al, 2000). GON-1 function is essential for the motility of DTCs and expansion of gonad arms, although the tissue distribution of GON-1 is not known (Blelloch and Kimble, 1999). Although lacking TS motifs, MIG-17 apparently belongs to the ADAMTS family based on significant homology between its MP, DI and PLAC (protease and lacunin) domains (of which the last was identified in this study) and those found in ADAMTS proteins (Nishiwaki et al, 2000). MIG-17 is secreted from body wall muscle cells and localizes to the gonadal basement membrane; it is required for directed DTC migration rather than motility per se (Nishiwaki et al, 2000). Figure 1.Tissue-specific expression of a membrane-anchored MIG-17. (A) The gonad morphology and the phases of DTC migration in wild-type hermaphrodites. (Upper) The gonad is shown in blue. Ventral and dorsal body wall muscles are shaded. Unshaded part between dorsal and ventral muscles corresponds to the hypodermis. (Lower) The phases of DTC migration are shown by arrows. (B) Abnormal gonad morphogenesis in mig-17 mutants. (C) (Upper) Domain structure and potential N-glycosylation sites of MIG-17. The circled numbers (1–9) indicate potential N-glycosylation sites (N-X-S/T). 1, N52; 2, N65; 3, N123; 4, N172; 5, N183; 6, N189; 7, N218; 8, N219; 9, N350. (Lower) Sequence alignment of the PLAC domains of MIG-17 and human ADAMTS-10. Identical and homologous amino acids are shown in black and gray, respectively. (D) A membrane-anchored MIG-17 can rescue mig-17 mutants when expressed in DTCs. The ratios of normal DTC migration are shown in bar graphs with the mean±s.e.m. For the animals carrying lag-2 promoter-driven transgenes, those with GFP expression in DTCs were scored. P-values from Fisher's exact test are shown for some combinations. n=120. DI, disintegrin domain; PLAC, protease and lacunin domain; MP, metalloprotease domain; Pro, prodomain; SP, signal peptide. Download figure Download PowerPoint MIG-17 offers an excellent model to study the molecular behavior and function of ADAMTSs during organ morphogenesis. In the present study, we used MIG-17 transgenes having mutations in the domains and glycosylation sites to investigate the regions in MIG-17 that are responsible for its localization and function in vivo. Using embryonic cell cultures, we found that mutant MIG-17 proteins are secreted with kinetics similar to that of the wild-type protein. We showed that MIG-17 is secreted as a proform and that the glycosylated prodomain plays an important role in targeting MIG-17 to the gonad. MIG-17 appears to be converted from the proform to the mature form via intramolecular autocatalytic activity, a process that is essential for MIG-17 to control DTC migration. Our findings provide a model of the action of ADAMTS proteases during organ formation and shed new light on the function of prodomains in targeting ADAMTSs to specific tissues or cells. Results MIG-17 is primarily required on the surface of DTCs The wild-type hermaphrodite gonad consists of two U-shaped arms formed by directed migration of DTCs. In mig-17 mutants, such U-shaped gonad morphogenesis is not achieved, and the gonad arms become deformed because of the aberrant migration of DTCs (Figure 1A and B). The domain structure of MIG-17 is shown in Figure 1C. We found that the C-terminal domain corresponds to the PLAC domain shared by several ADAMTSs. The PLAC domain of MIG-17 is most similar to that of human ADAMTS-10 (Somerville et al, 2004a) (Figure 1C). MIG-17 is secreted from the body wall muscle cells and localizes to the gonadal basement membrane shortly after the first turn of the DTCs (Nishiwaki et al, 2000). As ectopic expression of MIG-17 by the lag-2 promoter, which drives expression in DTCs, can rescue the DTC migration defects in mig-17 mutants, it has been assumed that MIG-17 localization on the DTC surface is important for its function (Nishiwaki et al, 2000). However, because MIG-17 is a secreted protein, it is still possible that MIG-17 produced by DTCs diffuses to its natural target tissues where it controls DTC migration. To examine whether MIG-17 is actually required on the DTC surface, we constructed a membrane-bound MIG-17 using the transmembrane domain of integrin-α, INA-1 (Baum and Garriga, 1997). When we expressed this MIG-17 construct tagged with GFP (MIG-17-TM-GFP) in body wall muscles using the endogenous promoter, the DTC migration defects of mig-17(k174) null mutants were weakly rescued for anterior DTCs, whereas they were mostly unrescuable for posterior DTCs. On the other hand, the expression of the same construct using the lag-2 promoter efficiently rescued both the anterior and posterior DTC migration defects, although the rescue was weaker than that achieved using MIG-17-GFP without a transmembrane domain (Figure 1D). These results suggest that the DTC surface is the primary site of action for MIG-17. The weaker rescue by the transmembrane form compared with the secreted form may suggest that anchoring to the plasma membrane weakly impairs the activity or mode of localization of MIG-17 in the gonadal basement membrane. N-glycosylation of the prodomain is essential for MIG-17 localization to the gonad mig-23 encodes a Golgi nucleoside diphosphatase required for protein glycosylation. We previously showed that mutations in mig-23 prevent MIG-17 localization to the gonad because of its defective glycosylation (Nishiwaki et al, 2004). However, the function of each glycan modification in MIG-17 remains unknown. MIG-17 contains six potential N-glycosylation sites (N-X-S/T) in the prodomain, three in the MP domain, but none in the DI and PLAC domains (Figure 1C). To address the roles of N-glycosylation in MIG-17, we mutated the MIG-17 N-glycosylation sites. The asparagine residues in these sites were changed to glutamines in various combinations (Figure 2A), thereby prohibiting N-glycosylation, and these MIG-17 mutants were expressed as GFP fusion proteins. Figure 2.Requirement of N-glycosylation for MIG-17 localization and function. (A) Glycosylation mutant constructs. Only the intact potential glycosylation sites are indicated. All asparagines of potential N-glycosylation sites were changed to glutamines in MIG-17(ΔGly1–9)-GFP: N52Q (AAT to CAA), N65Q (AAC to CAA), N123Q (AAT to CAA), N172Q (AAT to CAA), N183Q (AAC to CAA), N189Q (AAT to CAA), N218Q (AAC to CAA), N219Q (AAT to CAA) and N350Q (AAT to CAA). (B) Confocal (upper) and Nomarski (lower) images of wild-type hermaphrodites expressing MIG-17-GFP (left) or MIG-17(ΔGly1–9)-GFP (right). The boundaries of the gonads are depicted by a dotted line in the Nomarski images. Lateral views of posterior gonads. Dorsal to the top, anterior to the left. The gonadal localization of MIG-17 can be detected by linear GFP fluorescence between the proximal and distal gonad arms in animals expressing MIG-17-GFP (arrowhead). The strong dorsal and ventral GFP fluorescence corresponds to expression of transgenes in the body wall muscles (arrows). Punctate fluorescence outside the gonads is gut autofluorescence. Scale bar, 20 μm. (C) Western blot analysis. The lysates from wild-type worms expressing MIG-17-GFP or MIG-17(ΔGly1–9)-GFP were immunoblotted with anti-GFP. Pro- and mature forms are shown by arrows and arrowheads, respectively. The band of about 150 kDa in the MIG-17(ΔGly1–9)-GFP lane is often detected even in non-transgenic worms and probably due to nonspecific binding of the secondary antibody. (D) Requirement for glycosylation of MIG-17 in MIG-17 localization and function. Data for DTC migration are shown as in Figure 1D (n=120). Percentage of posterior gonads localized with GFP is indicated as the localization score on the right (n=20). Download figure Download PowerPoint We examined their localization to the gonad surface (gonadal basement membrane) in the wild-type background by confocal microscopy and their ability to rescue DTC migration defects in mig-17(k174) null mutants. Although the wild-type MIG-17-GFP efficiently localized to the gonad (90% of the gonads), the mutant protein that was disrupted at all nine potential glycosylation sites (MIG-17(ΔGly1–9)-GFP) failed to localize to the gonad (5% of the gonads) (Figure 2B and D). Because the lack of glycosylation could affect MIG-17-GFP secretion, we assessed whether MIG-17(ΔGly1–9)-GFP could be secreted from the muscle cells by examining endocytotic uptake of GFP fusion proteins by coelomocytes, which are scavenger cells in the body cavity (Fares and Greenwald, 2001a, 2001b). GFP fluorescence was observed in the coelomocytes in animals expressing MIG-17(ΔGly1–9)-GFP as well as those expressing MIG-17-GFP (data not shown). As discussed in a later section, the efficiency of secretion of the fusion protein was only slightly affected in MIG-17(ΔGly1–9)-GFP-expressing cells. Western blot analysis revealed that MIG-17(ΔGly1–9)-GFP migrates significantly faster than MIG-17-GFP, consistent with the idea that MIG-17 contains multiple N-glycosyl chains (Figure 2C). These results indicate that MIG-17 lacking N-glycosylation is secreted from the body wall muscle cells into the body cavity, and that N-glycosylation is essential for MIG-17 localization to the gonad. In contrast to the mostly normal DTC migration in wild-type worms, about 80% of the anterior or posterior gonads exhibited abnormal migration in mig-17(k174) mutants (Figure 2D). When MIG-17-GFP was expressed in the mig-17(k174) mutant, the migration defects were mostly rescued in both anterior and posterior gonads. When MIG-17(ΔGly1–9)-GFP was expressed in mig-17 mutants, although the migration defects of anterior DTCs were weakly rescued, the defects of the posterior DTCs were not (Figure 2D). Therefore, posterior DTC migration is strongly dependent on N-glycosylation of MIG-17. To determine which N-glycosylation site(s) (i.e., in the prodomain or MP domain) is important for gonadal localization of MIG-17, we constructed MIG-17(ΔGly1–6)-GFP, which lacks glycosylation in the prodomain, and MIG-17(ΔGly7–9)-GFP, which lacks glycosylation in the MP domain (Figure 2A). Although MIG-17(ΔGly7–9)-GFP localized to the wild-type gonad, MIG-17(ΔGly1–6)-GFP failed to do so (Figure 2D and Supplementary Figure S1). The mutant proteins having only either the first or last three glycosylation sites of the prodomain also failed to localize (Figure 2D). When these mutant constructs were expressed in mig-17 mutant animals, only MIG-17(ΔGly7–9)-GFP rescued the DTC migration defect (Figure 2D). These mutant proteins migrated faster than wild-type MIG-17 in Western blots (Supplementary Figure S2A). Examination of individual N-glycosylation sites revealed that the first, second, fourth and sixth sites in the prodomain are especially important for MIG-17 function as well as for localization (Supplementary Figure S3, Western blot in Supplementary Figure S2B). These results indicate that N-glycosylation of the prodomain is crucial for gonadal localization of MIG-17, whereas glycosylation of the MP domain has a minor role in this process. Prodomain glycosylation is required for MIG-17 localization but not for control of DTC migration We have shown that N-glycosylation of the prodomain is essential for MIG-17 localization and function. It is not clear, however, whether N-glycosylation is actually required for MIG-17 activity to control DTC migration, because the failure to localize appropriately precludes MIG-17 function on the gonad surface. Therefore, we expressed MIG-17(ΔGly1–6)-GFP or MIG-17(ΔGly1–9)-GFP in DTCs using the lag-2 promoter. Surprisingly, we found that both of these mutant proteins significantly rescued mig-17 mutants (Figure 2D). These results indicate that prodomain glycosylation is essential to recruit MIG-17 to the gonad surface but that it is dispensable for DTC migration after MIG-17 is localized to the gonad. Non-N-glycosylated MIG-17 probably has normal enzymatic activity against the physiological substrate required for DTC migration. A serine protease, matriptase, is also reported to retain normal substrate specificity after removal of N-linked polysaccharides (Ihara et al, 2004). Functions of MIG-17 domains Using transgenes mutated for each domain of MIG-17, we previously reported that the prodomain, DI domain and enzymatic activity are involved in MIG-17 localization to the gonad and that all domains are required for MIG-17's ability to control DTC migration (Nishiwaki et al, 2000). These analyses were qualitative, however, and thus we quantitatively re-evaluated the same constructs and several additional constructs (Figure 3A). The PLAC domain was completely dispensable for localization (Figure 3B). Although deletion of the DI domain (MIG-17(ΔDI)-GFP) or loss of proteinase activity (MIG-17(E303Q)-GFP) substantially weakened the GFP signal on the gonad surface, the signal was still detected in about half of the gonads examined. Deletion of the prodomain (MIG-17(ΔPro)-GFP) completely abolished localization. We generated MIG-17 mutant constructs D79N and G292E corresponding to the mig-17 alleles k135 and k176, which encode missense mutations within the pro- and MP domains, respectively, and have strong DTC migration defects similar to mig-17(k174) (Figure 3A). MIG-17(D79N)-GFP failed to localize, whereas MIG-17(G292E)-GFP localized normally (Figure 3B). Although these results raised the possibility that GFP fused with the prodomain alone (MIG-17(Pro1–209)-GFP) could localize to the gonad, this was not the case. The GFP fluorescence in coelomocytes was observed at similar levels in all transgenic animals for mutant constructs as well as in those for the wild-type construct (data not shown). We observed that secretion efficiency was only slightly affected in MIG-17(ΔPro)-GFP-expressing cells, as discussed later. These results indicate that the prodomain is very important for MIG-17 localization to the gonad, but it is not sufficient. When these constructs were introduced into mig-17 mutants, they all exhibited very little rescue activity—especially for posterior DTC migration abnormalities (Figure 3B). Figure 3.Requirement of domains for localization and function of MIG-17. (A) MIG-17 mutant constructs. Deleted regions are shown as crosshatched boxes. The amino-acid positions of the N and C termini of the deleted regions are indicated. Positions for amino-acid changes for MIG-17(E303Q)-GFP, MIG-17(D79N) and MIG-17(G292E) are shown. (B) mig-17 rescue experiments. Data for DTC migration are shown as in Figure 1D (n=120). Asterisks indicate P<0.001 in Fisher's exact test against the score of mig-17(k174)Ex[mig-17∷GFP]. Localization scores are shown on the right (n=20). Download figure Download PowerPoint Prodomain processing requires the autocatalytic activity of MIG-17 Most of the ADAMs and ADAMTSs reportedly are cleaved between the prodomain and MP domain as they progress through the secretory pathway. Prodomain processing can be recapitulated in vitro with cell lysates in some cases (Schlomann et al, 2002). Therefore, we examined prodomain processing of various MIG-17-GFP constructs using worm lysates. Western blotting of extracts of wild-type animals expressing MIG-17-GFP showed mostly the proform with only a small amount of the mature form (Figure 2C). However, when the extracts were incubated at room temperature, the amount of the mature form gradually increased (Figure 4A, left panel). In contrast, the protease-deficient form, MIG-17(E303Q)-GFP, existed only as a proform and was never processed to maturity during incubation, suggesting that autocatalytic activity is necessary for conversion to the mature form and that endogenous wild-type MIG-17 cannot process the mutant MIG-17(E303Q)-GFP (Figure 4A, right panel). Therefore, the prodomain of MIG-17 is probably removed via an intramolecular autocatalytic activity, a conclusion that is supported by the fact that the serine and cysteine protease (but not MP) inhibitors aprotinin and leupeptin failed to inhibit the reaction (data not shown). Figure 4.Prodomain processing is essential for MIG-17 function in cell migration. (A) Processing of MIG-17 in vitro. The lysates from wild-type worms expressing MIG-17-GFP or MIG-17(E303Q)-GFP were incubated at room temperature for the indicated periods and immunoblotted with anti-GFP. (B) Mutant constructs of potential processing sites. (C) The extracts from wild-type animals expressing MIG-17(KK202LL)-GFP were analyzed as in (A). (D) Gonadal localization of MIG-17(KK202LL)-GFP. Scale bar, 20 μm. (E) mig-17 rescue experiments using the constructs in (B). Data for DTC migration are shown as in Figure 1D (n=120). The localization scores are shown on the right (n=20). (F, G) Processing of glycosylation mutants MIG-17(ΔGly7–9)-GFP and MIG-17(ΔGly1–6)-GFP in vitro. Experiments were preformed as in (A). The mature form of MIG-17(ΔGly1–6)-GFP can be detected after a 6-h incubation. Download figure Download PowerPoint Prodomain processing is essential for MIG-17 activity in controlling DTC migration Although the actual processing site of MIG-17 is not known, prodomain processing of ADAMTS proteases often occurs in the region of consecutive basic amino acids (Cal et al, 2001; Longpre and Leduc, 2004; Somerville et al, 2004b). To determine the processing site, we individually altered three sets of two consecutive basic residues, Arg162Arg163, Lys202Lys203 and Arg205Lys206, to leucines (Figure 4B). We introduced these mutant constructs into wild-type worms. Although the proteins carrying substitutions at Arg162Arg163 (MIG-17(RR162LL)-GFP) and Arg205Lys206(MIG-17(RK205LL)-GFP) were processed (data not shown), the protein carrying substitutions at Lys202Lys203 (MIG-17(KK202LL)-GFP) was not processed (Figure 4C). Therefore, it is likely that the processing occurs at or near Lys202Lys203. These three constructs localized to the gonad surface (Figure 4D and E) and, when expressed in mig-17 mutants, MIG-17(RR162LL)-GFP and MIG-17(RK205LL)-GFP rescued the DTC migration defects, whereas the rescue was severely impaired in mutants expressing MIG-17(KK202LL)-GFP (Figure 4E). These results indicate that MIG-17 prodomain processing is not required for localization; however, the processing is essential for the function of MIG-17 to control the migration of DTCs. The presence of the prodomain thus seems to inhibit the ability of MIG-17 to promote DTC migration. We examined whether other mutant constructs that localized or weakly localized to gonads but had very weak rescuing activity could be converted to the mature protease in vitro. MIG-17(D79N)-GFP, MIG-17(ΔDI)-GFP, MIG-17(ΔPLAC)-GFP and MIG-17(G292E)-GFP failed to be converted to the mature form (data not shown). Therefore, these mutations affect prodomain processing. In addition, we also assessed the effect of prodomain processing in the N-glycosylation mutants of MIG-17. MIG-17(ΔGly7–9)-GFP, which rescued mig-17, was processed normally (Figure 4F). The processing of MIG-17(ΔGly1–6)-GFP, which rescued mig-17 when expressed in DTCs but not when expressed in muscles, was very slow, but the mature form accumulated over time (Figure 4G). These results are consistent with the idea that prodomain processing is essential for MIG-17 activity in controlling DTC migration. To examine whether prodomain processing is required for MIG-17 control of DTC migration in vivo, we tried to change the potential autocatalytic processing site of MIG-17 into the recognition site of furin, a Golgi enzyme that acts in prodomain processing of various proteases. The KLRK residues from 203 to 206 were substituted with RRRR (Figure 4B). When we expressed MIG-17(KLRK203RRRR)-GFP in the muscle cells, it successfully localized to the gonad but failed to rescue mig-17. Surprisingly, however, it efficiently rescued mig-17 when expressed in DTCs using the lag-2 promoter (Figure 4E). Western blot analysis revealed that prodomain processing of the mutant protein was much more efficient than autocatalytic processing although unprocessed proforms still existed (Supplementary Figure S4). We speculate that the prodomain of MIG-17(KLRK203RRRR)-GFP is processed by furin in the Golgi and that the mature MIG-17-GFP can function in DTC migration when it is secreted from DTCs but cannot when secreted from muscles. We therefore suggest that only unprocessed MIG-17-GFP secreted from muscles can localize to the gonad owing to the presence of the prodomain, which cannot be processed autocatalytically. To examine the activity of prodomain mutants, we expressed MIG-17(ΔPro)-GFP, MIG-17(D79N)-GFP and MIG-17(D79N, KLRK203RRRR)-GFP under the control of the lag-2 promoter. We found that all these constructs rescued mig-17 very weakly (Figure 4E). Western blot analysis revealed that MIG-17(D79N)-GFP was not processed, whereas MIG-17(D79N, KLRK203RRRR)-GFP was indeed processed (Supplementary Figure S4), suggesting that the mature form generated from MIG-17(D79N, KLRK203RRRR)-GFP is defective in controlling leader cell migration. MIG-17 lacking N-glycosylation or the prodomain is secreted almost normally To analyze quantitatively the secretion efficiencies of wild-type and mutant GFP-fusion proteins, we prepared primary cell cultures from transgenic embryos (Christensen et al, 2002). In the cell culture, muscle cells expressing MIG-17-GFP fusion proteins could be detected with fluorescence microscopy (Figure 5A). Sampling of culture medium after 24, 48 and 72 h in serum-free culture followed by Western blotting revealed gradual accumulation of secreted MIG-17-GFP in pro- and mature forms in the medium (Figure 5B). MIG-17(ΔGly1–9)-GFP similarly accumulated over time, although much more proform than mature form accumulated (Figure 5C). This is consistent with the slow processing observed in MIG-17(ΔGly1–6)-GFP (Figure 4G). MIG-17(ΔPro)-GFP also progressively accumulated in the medium (Figure 5D). Because it was possible that accumulation of MIG-17-GFP fusion proteins in the culture media could be due to leakage of these proteins from dead cells rather than secretion, we examined the primary culture from embryos expressing a membrane-bound construct, MIG-17-TM-GFP. We observed no accumulation of this protein in the medium (Figure 5E). It seems that MIG-17-TM-GFP can be efficiently processed into the mature form while retained at the cell surface. Therefore, the MIG-17-GFP proteins detected in the media were probably secreted. We assessed the secretion efficiency by determining the ratio of MIG-17-GFP proteins found in the medium to those retained in the cells. As shown in Figure 5F, the kinetics of secretion for these three constructs were almost similar except that a slight reduction in secretion was detected in cells expressing MIG-17(ΔGly1–9)-GFP or MIG-17(ΔPro)-GFP after 72 h. These results suggest that deletion of N-glycosylation sites or the prodomain does not significantly affect the secretion of MIG-17-GFP proteins. Figure 5.Secretion of MIG-17 from primary culture cells. (A) A typical culture of C. elegans embryonic cells 4 days after plating. Nomarski (upper) and combined Nomarski and fluorescence (lower) images of MIG-17-GFP-expressing primary culture cells. (B–E) Secretion of MIG-17-GFP, MIG-17(ΔGly1–9)-GFP, MIG-17(ΔPro)-GFP and MIG-17-TM-GFP into the media. The culture media were sampled at the indicated time points and cell lysates were prepared from 72-h cultures. The samples were immunoblotted with anti-GFP. The same set of experiments (from culture to immunoblot) was independently performed twice. The ratios of mature form to the sum of pro- and mature forms are shown for some lanes as the mean±s.e.m. The asterisk indicates that the rate of conversion from the proform to mature form was significantly slower in MIG-17(ΔGly1–9)-GFP-expressing cells compared with MIG-17-GFP-expressing cells (Student's t-test; P<0.05). The smeared band migrating slightly faster than the mature form (open arrowhead) in (E) appears to be partially degraded proteins. (F) Kinetics of secretion of MIG-17-GFP fusion proteins. The ratios of secreted MIG-17-GFP fusion protein to MIG-17-GFP fusion protein retained in the cell lysates were plotted against sampling times. For MIG-17-GFP, MIG-17(ΔGly1–9)-GFP and MIG-17-TM-GFP, the intensitiesy of the bands for the pro- and mature forms were summed and used for calculation. The error bars represent the mean±s.e.m. Download figure Download PowerPoint MIG-17 is secreted and localizes as a proform To understand the distribution of endogenous MIG-17, we generated an antibody against its prodomain. This anti-MIG-17 prodomain antibody recognized the proform but not the mature form of MIG-17-GFP in a Western blot (Figure 6A and B). When we immunostained cross-sections of wild-type and mig-17 mutant animals, we detected specific signals only in the wild-type specimens. The signal was detected on the surface of the gonad and within the gonad. In addition, we detected somewhat weaker signals at the surfaces of the intestine and hypodermis, as well as in the intestinal lumen (Figure 6C, D and G). Because MIG-17-GFP is secreted from the muscle cells and localized to the gonad surface (Nishiwaki et al, 2000), these results strongly suggest that MIG-17 is secreted as a proform and diffuses to various tissues. It is likely that a portion of the MIG-17 population is internalized in these tissues. The localization of the signal at the intestinal lumen and the apical surface of the hypodermis suggests that some MIG-17 is internalized and transported to these sites. To confirm the observed tissue distribution of wild-type and mutant MIG-17-GFP proteins, we also immunostained cross-sectio
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