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Identification of a novel ADAMTS9/GON-1 function for protein transport from the ER to the Golgi

ADAMTS9 is required for protein transport from the ER to the Golgi, which is dependent on the C-terminal GON domain but not on protease activity. This novel function of the GON domain is conserved in both C. elegans (GON-1) and mammalian cells (ADAMTS9). The GON domain acts inside the ER but not outside the cells.


INTRODUCTION
The A disintegrin-like and metalloprotease with thrombospondin type I motif (ADAMTS) family comprises secreted zinc metalloproteases with a precisely ordered modular organization that includes at least one thrombospondin type I repeat (Kuno et al., 1997;Hurskainen et al., 1999;Clark et al., 2000). ADAMTS9 localizes extracellularly or on the cell surface and cleaves large, aggregating proteoglycans such as aggrecan and versican (Somerville et al., 2003). Similar to most zymogens, the ADAMTS9 protease domain contains an N-terminal propeptide. The propeptide is believed to have a role in maintaining enzyme latency, and excision of the propeptide activates ADAMTS9. The ADAMTS9 propeptide is processed by furin at the cell surface (Somerville et al., 2003) and is necessary for localization to the cell surface (Koo et al., 2007). ADAMTS9 mRNA is widely expressed, especially during embryonic development in both humans and mice (Somerville et al., 2003;Jungers et al., 2005).
In humans, an ADAMTS9 gene variant in the 5′-upstream region (rs4607103) is associated with type 2 diabetes (Grarup et al., 2008;Zeggini et al., 2008), and this allele results in reduced insulin-stimulated glucose uptake (Boesgaard et al., 2009). The molecular mechanisms underlying the effect of ADAMTS9 on the peripheral insulin action and risk of diabetes, however, are unknown. In mice, an ADAMTS9-null allele is early embryonic lethal (Silver et al., 2008), suggesting that ADAMTS9 plays an essential role in organism survival, although the molecular mechanisms of ADAMTS9 involvement during embryogenesis are unknown. GON-1 is the Caenorhabditis elegans orthologue of ADAMTS9 and is required for distal tip cell (DTC) migration during gonad morphogenesis. GON-1 contributes to degradation of the basement membrane or the processing served in DTCs of almost all of the transgenic animals treated with gon-1(RNAi) or in the gon-1(tm3146) background (see Figure 1C, middle and right, and Figure 3A later in the paper). of extracellular cues required for cell migration by its protease activity . It is of interest that membranes accumulate within the cytoplasm in DTC of gon-1 mutants . This phenotype is not predicted by the known protease functions of GON-1 and ADAMTS-family proteases (Porter et al., 2005). Thus these previous reports suggest that  have an additional function.
Here we report on the novel function of ADAMTS9 and GON-1, which are required for the protein secretory pathway from the endoplasmic reticulum (ER) to the Golgi in both C. elegans and human cells. It is surprising that this novel function depends on the GON domain without protease activity, and the GON domain plays a role in the ER but not extracellularly. The GON domain exists at the C-terminus of ADAMTS9, ADAMTS20, and GON-1 and in a number of GON-domain proteins in metazoans, suggesting that its role has broad biological significance. This is the first report of the GONdomain function, which is important for protein transport from the ER to the Golgi.

RESULTS
Gonad proliferation defects in a gon-1 mutant are associated with abnormalities in ER structure and function and the ER stress response A member of the ADAMTS-like metalloprotease family is encoded by gon-1, which is expressed in DTCs and body wall muscle cells and functions in extracellular matrix (ECM) remodeling in C. elegans (Jones and Riley, 2005). Depletion of GON-1 results in gonad proliferation defects and aberrant membrane morphology in hermaphrodite DTCs . We confirmed this phenotype using RNA interference (RNAi) of gon-1 [gon-1(RNAi)] in wild-type animals and a gon-1 mutant (tm3146) ( Figure 1A and Supplemental Figure S1). gon-1(tm3146) harbors a deletion that removes the protease domain, and a termination codon is introduced by a neighboring short insertion (Supplemental Figure S2).
We examined possible changes in the organelle structures of DTC in the gon-1 reduction-of-function background to gain insights into the accumulation of the intracellular membrane. We generated transgenic strains that harbor a DTC-specific, lag-2 promoter-driven cytochrome b5-a putative ER protein-fused to Venus. As expected, a fluorescence signal was observed around the nuclei of the DTCs in wild-type animals (Figure 1B,left). Treatment of the same transgenic strain by gon-1(RNAi) or introduction of the same transgene into the gon-1(tm3146) background resulted in a large and deformed green fluorescent protein (GFP)-positive structure in DTCs. The deformed fluorescence signals were observed as a few circular structures that were closely apposed to each other. The circular structures were also visible in differential interference contrast (DIC) images (Figure 1B,arrowhead). This phenotype might correspond to the extensive expansion of the membrane structure observed by electron microscopy (see Figure 1B, middle and right, Figure 3A later in the paper, and Supplemental Figure S1). The same results were observed when Venus-SP12 was used as another ER marker (Poteryaev et al., 2005;(Supplemental Figure S3). The expression patterns of other organelle markers were not greatly changed by gon-1(RNAi) (Supplemental Figure S4). An ER-containing autophagosome (ERA)-like structure was also observed by electron microscopy (Supplemental Figure S1). ERA-like structures were reported to appear upon ER dysfunction (Bernales et al., 2006). We next evaluated whether protein secretion was impaired in gon-1(RNAi) because the ER structure was abnormal. The protein secretion activity of DTCs was examined based on the distribution of secreted soluble Venus (ssVenus) expressed in DTCs (Fares and Greenwald, 2001). Accumulation of Venus fluorescence was ob-FIGURE 1: gon-1(tm3146) has an aberrant morphology of the ER and secretion defects. (A) In gon-1 mutants and gon-1(RNAi) animals, gonadal arm extension did not occur. Arrowheads indicate the position of the tip of the gonad. The schematic drawings (right) indicate the morphology of the gonad. Scale bar, 100 μm. (B) ER morphology analyzed by an ER marker, cytochrome b5-Venus. Confocal fluorescence images drastically changed in animals treated with gon-1(RNAi) and gon-1(tm3146) mutants (top). Arrowheads indicate abnormal ER morphology. Bottom, the differential interference contrast (DIC) images. Scale bar, 5 μm. (C) Protein secretion from DTC was blocked in animals with gon-1(RNAi) and gon-1(tm3146) mutants (top). Bottom, the DIC images. Scale bar, 50 μm. (D) Transportation analyzed by a plasma membrane protein marker. Confocal fluorescence images changed in animals treated with gon-1(RNAi) and gon-1(tm3146) mutants (top). Bottom, the DIC images. Scale bar, 5 μm.
We then evaluated whether gon-1 is necessary for the transportation of a plasma membrane protein and a glycosylphosphatidylinositol (GPI)-anchored protein. We generated transgenic worms expressing a lag-2 promoter-driven enhanced GFP (EGFP) fused with the transmembrane (TM) domain of T01F10.1a (TM-EGFP) as a plasma membrane protein and yellow fluorescent protein (YFP)-GPI as a GPI-anchored protein. In wild-type animals, TM-EGFP and YFP-GPI localized to the plasma membrane ( Figure  1C, left, and Supplemental Figure S5A, left). Punctate accumulation of TM-EGFP was observed in DTCs when the transgenic animals were treated by gon-1(RNAi) or in the gon-1(tm3146) background ( Figure 1D, middle and right). The YFP-GPI signal accumulated in DTCs when transgenic animals were treated with gon-1(RNAi) (Supplemental Figure S5A, right). ER dysfunction in body wall muscle cells in the gon-1(tm3146) background was examined by evaluating protein secretion in transgenic worms harboring arIs37, which expressed and secreted soluble GFP from body wall muscles to the pseudocoelom. The secreted GFP in the pseudocoelom was endocytosed and accumulated in coelomocytes in wild-type animals (Supplemental Figure S5B, left, arrowhead), as previously reported (Fares and Greenwald, 2001). On the other hand, bright fluorescence was observed in the body wall muscle cells of gon-1(tm3146) mutants (Supplemental Figure S5B, right). We also examined the distribution of TM-EGFP expressed in body wall muscle cells. In wild-type animals, TM-EGFP mainly localized to the plasma membrane (Supplemental Figure S5C, left). In contrast, depletion of gon-1 led to the punctate accumulation of TM-EGFP in body wall muscle cells (Supplemental Figure  S5C, right), indicating that GON-1 is also required for TM-EGFP transport in body wall muscle cells.
The BiP protein, an ER-stress sensor, is induced when ER functions are impaired in mammalian cells. To determine whether the homologue in the nematode (hsp-4) is also induced in gon-1(RNAi)treated animals, we used a transgenic allele zcIs4 (Calfon et al., 2002) harboring a transcriptional fusion reporter of phsp-4::gfp. In wild-type animals, GFP expression was not observed in DTCs (Supplemental Figure S6A, left). By contrast, gon-1(RNAi) induced GFP expression in DTCs (Supplemental Figure S6A, right).
To examine the relationship between ER function and gonad proliferation and migration, we depleted oligosaccharyltransferase (T12A2.2) using RNAi. Oligosaccharyltransferase is essential for Nlinked glycosylation in the ER (Dempski and Imperiali, 2002). Therefore, knockdown of this gene should mimic the treatment of animals with tunicamycin, which is a chemical inhibitor of N-linked glycosyla-tion and a typical ER stress-inducing reagent. Gonad proliferation was also highly disturbed by RNAi knockdown of the T12A2.2 gene in N2 worms, as in gon-1-mutant animals (Supplemental Figure S7). These results suggest that down-regulation of ER function accounts for some of the gonadal defects.
These findings suggest that gon-1 functions both inside and outside of the cell. On the basis of these data, we propose that localization of the GON domain in the ER is important for intracellular function and that localization of gon-1, both inside and outside of the cell, is important for gonad proliferation.
Deformation of ER structure, inhibition of protein secretion, and frame-switch splicing of xbp-1 are induced after GON domain inactivation We observed the time course of the morphological changes in the ER and the inhibition of protein secretion by chromophore-assisted light inactivation of the GON domain (Bulina et al., 2006). We created a light-inactivatable fusion protein of the KillerRed and GON domain by substituting Venus in gon-1(sig_Venus_GON) with Kill-erRed. We generated transgenic animals that expressed lag-2 promoter-driven gon-1(sig_KillerRed_GON) in the gon-1(tm3146) mutant background ( Figure 4A). This transgene also rescued the ER morphology, accumulation of ssVenus, and splicing of xbp-1 in DTCs in the absence of green-light illumination. When the animals are illuminated with green light, the GON domain fused to KillerRed is inactivated (Bulina et al., 2006). Thus we could inactivate the GON domain by illuminating the animals with green light.
We generated transgenic animals expressing lag-2 promoterdriven gon-1(sig_KillerRed_GON) and an ER marker in gon-1(tm3146) mutants and evaluated ER morphology using fluorescence microscopy after green-light irradiation for 0, 2, 5, 10, and 24 h. Light-induced inactivation of the GON domain affected the ER morphology after 24 h of green-light exposure. In contrast, in the dark control, abnormal ER morphology was rarely observed (Figure 4, B and C).
In transgenic animals expressing lag-2 promoter-driven gon-1(sig_KillerRed_GON) and ssVenus in gon-1(tm3146) mutants, ssVenus accumulation in the DTCs was examined after irradiation with green light for 0, 2, 5, 10, and 24 h. Accumulation of ssVenus induced by light inactivation of the GON domain was observed after irradiation for 2 h. In the dark control, there was only slight accumulation of ssVenus (Figure 4, D and E).

Depletion of ADAMTS9 in a mammalian cell line inhibits transport from the ER to the Golgi
To determine whether the gon-1-like protein function is conserved in mammalian cells, we performed small interfering RNA (siRNA)-mediated knockdown of ADAMTS9, a human homologue of GON-1, in HEK293 cells. Staining with an ER-Tracker showed that ADAMTS9 depletion induced deformation of the ER morphology ( Figure 5A). ER-Tracker-positive globular structures were observed in ADAMTS9 siRNA-treated cells but rarely in the control siRNA-treated cells. To confirm the abnormal ER structures in the ADAMTS9 siRNA-treated cells, we examined the ER structure with electron microscopy. We observed tightly stacked whirling membrane patterns in ADAMTS9-depleted cells but not in the control siRNA-treated cells (Supplemental Figure S10).
Vesicular transport was observed using the temperature-sensitive vesicular stomatitis virus glycoprotein (VSVG; ts045) fused to GFP. In this experimental system, VSVG-GFP first accumulates in the ER when cultured at 40°C, and when the temperature is shifted to 32°C, the molecules are translocated from the ER to the plasma membrane through the Golgi. Localization of VSVG-GFP was evaluated by immunostaining with an anti-GM130 antibody or an anti-KDEL antibody. As expected, in the cells treated with control siRNA or ADAMTS9 siRNA, when cultured at 40°C, VSVG-GFP fluorescence localized to the ER, as shown by colocalization of anti-KDEL antibody staining (Supplemental Figure S11), but not to the Golgi, as shown by colocalization of anti-GM130 antibody staining ( Figure 5B). After 20 min at the permissive temperature, in the cells treated with control siRNA, VSVG-GFP fluorescence localized to the Golgi in 76% of the cells, based on the colocalization of anti-GM130 antibody staining ( Figure 5, B and C), but not in the ER, based on the colocalization of anti-KDEL antibody staining (Supplemental Figure S11). VSVG-GFP fluorescence localized to both the Golgi and the ER in 22% of the cells. At later time points, the VSVG-GFP was eventually translocated to the plasma membrane (unpublished data). In contrast, in ADAMTS9 siRNA-treated cells, 20 min after transfer to the permissive temperature, VSVG-GFP fluorescence localized to the Golgi in 18% of the cells and to both the Golgi and the ER in 27% of the cells. VSVG-GFP fluorescence localized to the ER in 55% of the cells, as shown by colocalization of anti-KDEL antibody staining ( Figure 5C and Supplemental Figure S11). VSVG-GFP fluorescence did not colocalize with anti-GM130 antibody immunoreactivity in 55% of the cells ( Figure 5B). These results indicate that ADAMTS9 knockdown caused the accumulation of VSVG-GFP within the ER 20 min after shifting the temperature to 32°C in HEK293 cells ( Figure 5, B and C, and Supplemental Figure S11).
To investigate other phenotypes in mammalian cells, we examined whether ADAMTS9 is required for the secretory function in HEK293 cells. Knockdown of ADAMTS9 caused the accumulation of GFP fused with the prolactin (PRL) secretory signal within the cells (Supplemental Figure S12). We also examined whether frame-switch splicing of XBP1 is observed in ADAMTS9 siRNA-treated mammalian cells. This was indeed the case in HEK293 cells (Supplemental Figure S13A). Although the cell morphology changed a little, To investigate whether the GON domain alone could rescue the ER-to-Golgi transport phenotype in HEK293 cells, as observed in C. elegans, we transfected VSVG-GFP and gon-1(sig_GON) into HEK293 cells treated with ADAMTS9 siRNA or control siRNA. In this becoming rounder ( Figure 5A), and too strong ER stress may cause apoptosis, we examined cell viability of ADAMTS9 siRNA-treated HEK293 cells. The cell viability was unchanged between control and ADAMTS9 siRNA-treated cells (Supplemental Figure S13B).  Figure S8. Domains are indicated as described in Figure 2A. (B) ER morphology of gon-1 mutants that expressed plag-2::gon-1(sig_KillerRed_GON) and plag-2::C31E10.7::Venus was observed at the indicated time points by confocal fluorescence microscopy after illumination with a green light (photoablation) or not (control). ER morphology changed dramatically between 10 and 24 h after irradiation, as shown by arrowheads. Scale bar, 5 μm. (C) Quantification of the results in B. The graph represents the combined results of six independent experiments (n > 100 for each experiment). Bars represent mean ± SE. (D) Accumulation of ssVenus within DTC in gon-1 mutants that expressed plag-2::gon-1(sig_KillerRed_GON) and plag-2::ssVenus was observed at the indicated time points by confocal fluorescence microscopy after illumination with a green light (photoablation) or not (control). ssVenus accumulation in DTC was observed after 2 h of irradiation. (E) Quantification of the results in D. The graph represents the combined results of five independent experiments (n > 90 for each experiment). Bars represent mean ± SE. ization of VSVG-GFP was evaluated. At 40°C, VSVG-GFP accumulated in the ER, as shown by colocalization of anti-KDEL antibody staining in 100% of the gon-1(sig_GON)-introduced and control siRNA-treated cells and in 100% of the gon-1(sig_GON)-introduced ADAMTS9 siRNAtreated cells (Figure 6, A and B, and Supplemental Figure S14). After 20 min at the permissive temperature, VSVG-GFP localized to the Golgi, as shown by colocalization of anti-GM130 antibody staining in 85% of the gon-1(sig_GON)-introduced and control siRNA-treated cells. After 20 min at the permissive temperature, VSVG-GFP localized to the Golgi in 83% of the gon-1(sig_ GON)-introduced and ADAMTS9 siRNAtreated cells (Figure 6, A and B). VSVG-GFP did not localize to the ER, as shown by colocalization of anti-KDEL antibody staining in these cells (Supplemental Figure S14). These observations support the idea that the GON domain, but not the protease activity, is required for cargo transport from the ER to the Golgi in HEK293 cells.
We next analyzed the subcellular localization of various GON-domain proteins in mammalian cells to determine protein structure-function relationship. To do this, we transfected gon-1(sig_GON_Venus), gon-1(sig_Venus_GON), ADAMTS9(sig_GON_ Venus), or ADAMTS9(sig_Venus_GON) into HEK293 cells and observed Venus fluorescence localization. When HEK293 cells were transfected with plasmids expressing gon-1(sig_Venus_GON) and ADAMTS9(sig_ Venus_GON), fluorescence was observed in the ER, as shown by colocalization with the calnexin immunoreactivity. In contrast, gon-1(sig_GON_Venus) and ADAMTS9(sig_ GON_Venus) localized diffusely throughout the cytoplasm (Supplemental Figure S15). These findings are similar to the localization of the GON domain in the nematode. Taken together, these observations of HEK293 cells were similar to the phenotypes found in the nematode.
Because cargo molecules accumulated in the ER, and ER stress was induced under the ADAMTS9-depleted condition, we evaluated whether secretory proteins are normally O-linked glycosylated in the Golgi and degraded normally by the proteasome system. We expressed α1-antitrypsin (AAT) to monitor secretion in HEK293 cells treated with or without ADAMTS9 siRNA and found that ADAMTS9 knockdown caused the accumulation of AAT within the cells and a reduction of AAT in the medium (Supplemental Figure S16, A-C), similar to the accumulation of the prolactin-GFP fusion protein described earlier. We used the enzyme endoglycosidase H (Endo H), which removes N-linked glycosylation, but not O-linked glycosylation, from proteins in the siRNA-plasmid transfection experiment, because the GON-domain nucleotide sequences are very different between human and C. elegans, the transfected siRNA should not knock down the GON domain mRNA from the rescue plasmid. These cells were stained with an anti-GM130 antibody or an anti-KDEL antibody, and local-FIGURE 5: Knockdown of ADAMTS9 inhibited ER to Golgi transport in mammalian cells. (A) The shrunken ER was observed in the cells treated with ADAMTS9 siRNA (arrowheads). The siRNA-treated cells were stained with an ER-tracker (left), and DIC images of the same field are shown (right). Scale bar, 20 μm. (B) VSVG-expressing cells were incubated for 16 h at 40°C and then shifted to 32°C. The cells were fixed at the indicated time points following transfer to the permissive temperature. The Golgi apparatus was labeled by an anti-GM130 antibody (middle). VSVG-GFP images were merged with those of the Golgi (right). VSVG-GFP transport from the ER to the Golgi was inhibited in the cells in which siRNA-mediated knockdown of ADAMTS9 was performed. Arrowheads indicate the shrunken ER in ADAMTS9-knockdown cells. Scale bar, 5 μm. (C) Quantification of the VSVG transports. The graph represents the combined results of four independent experiments (n > 50 for each experiment). Bars represent mean ± SE. secretory pathway. Endo H cleaved all the carbohydrate chains of AAT that remained within the cells in both control and ADAMTS9 siRNA-treated cells (Supplemental Figure  S16C, Cell). On the other hand, irrespective of siRNA treatment, secreted AAT was Endo H resistant due to the oligosaccharide modification that occurred in the Golgi apparatus (Supplemental Figure S16C, Medium). Thus glycosylation was not affected by ADAMTS9 siRNA treatment.
To examine whether ADAMTS9 is required for ER-associated degradation (ERAD) in the ER, we transfected an ATT variant, null Hong Kong (NHK), which fails to be transported to the Golgi and is degraded by ERAD (Hosokawa et al., 2003), in HEK293 cells. We examined the time course of the degradation of the NHK variant in a cycloheximide-chase experiment with or without ADAMTS9 siRNA. Degradation of the NHK variant did not differ between cells treated with ADAMTS9 siRNA and control siRNA (Supplemental Figure S16D). This finding suggested that degradation of AAT(NHK) by ERAD is not dependent on ADAMTS9. (Jones and Riley, 2005). The gon-1 gene has an essential role in gonad development in C. elegans . The present study revealed that C. elegans GON-1, an ADAMTS9 homologue, and human ADAMTS9 also have another function. GON-1 and ADAMTS9 are involved in the mechanisms of protein transport from the ER to the Golgi. In addition, the GON domain at the C-terminus of GON-1 and ADAMTS9 localizes to the ER, and the GON domain is important for ER functions.

Distal tip cells and body wall muscle cells express gon-1, which encodes a ADAMTSlike metalloprotease that functions in ECM remodeling
In C. elegans, down-regulation of gon-1 led to ER deformation and gonad proliferation defects, and these phenotypes were rescued by GON-domain expression without the protease domain in the ER of DTCs. To investigate whether the role of the GON domain in the ER of DTCs is sufficient for gonad proliferation, we compared transgenic animals harboring two our own observations. The GON domain is present in the most-Cterminal region in these proteins (Supplemental Table S1). These results are consistent with our finding that gon-1(sig_Venus_GON) had a higher rescue activity than gon-1(sig_GON_Venus). In addition, the presence of Venus in the C-terminus disrupted the localization of the GON domain in both C. elegans and mammalian cells. Thus the C-terminus of the GON domain most likely has an important role in its localization and eventually its function.
Newly synthesized secretory proteins are transported from the ER to the Golgi by COPII-coated vesicles. A large number of proteins, including coat proteins that help vesicle budding, proteins that regulate cargo export, and receptors that bind to secretory cargoes, are required for the biogenesis of COPII-coated vesicles. COPII-coated vesicles comprise the sec23/24 and sec13/31 complexes (Gurkan et al., 2006). Export of cargo proteins from the ER is mediated by sar-1, sec16, and sec12 (Gurkan et al., 2006). TFG-1 regulates cargo export from the ER (Witte et al., 2011). ERGIC-53, Emp24p, surf4, and TANGO1 function as cargo receptors (Nakamura et al., 1998;Muniz et al., 2000;Mitrovic et al., 2008;Nyfeler et al., 2008;Saito et al., 2009). These cargo receptors are believed to mediate cargo loading into COPII vesicles, and the cargo proteins are then exported from the ER to the Golgi. ERGIC-53 has a role in the transport of coagulation factors V and VIII, cathepsin C and Z, and AAT. Emp24p is involved in the transport of Gas1p, a GPI-anchored protein. Surf4 has a role in the transport of soluble proteins. TANGO1 has a role in the transport of collagen VII. These factors are all considered to be involved in transportation of limited types of cargoes. On the other hand, depletion of gon-1 led to accumulation of TM-EGFP, a TM protein, and a GPI-anchored protein, as well as secretory proteins such as ssVenus, suggesting that the GON domain acts on a variety of cargoes in the secretory pathway. Thus the function of the GON domain appears to differ from those of other transport factors. The amounts and types of cargoes to be transported differ among cell types, but mechanisms of substrate specificity and cell specificity involved in cargo export are not fully elucidated. Of interest, not all cells express GON-1 and ADAMTS9 in the nematode and human, but all cells have some mechanisms of transport from the ER to the Golgi. The dissimilarities among cell types that require the GON domain are not clear. Future studies are needed to elucidate the molecular mechanisms of the action of the GON domain in the ER.

Nematode strains
The deletion mutant strain of gon-1(tm3146) was obtained as described (Gengyo-Ando and Mitani, 2000). The deletion allele was identified by PCR amplification with the primers spanning the deleted region as described in Supplemental Table S2 and Supplemental Figure S2. gon-1(tm3146) was derived from the wild-type Bristol strain N2. Genetic crosses were carried out by the standard genetic protocols (Brenner, 1974).

Generation of transgenic strains
The transgenic strains used in this work are listed in Supplemental  Table S3. The sequences of the primers for construction of plasmids used for microinjection are listed in Supplemental Table S4. For expression of target proteins in DTC, the promoter region of lag-2 was amplified by PCR from the C. elegans genomic DNA using the primers of plag-2-FW and plag-2-RV and inserted into the pPD95.79 vector plasmid (a gift from A. Fire, Stanford University), giving rise to pPD95.79_plag-2. The GFP cDNA that was originally inserted in pPD95.79 was replaced with Venus at KpnI and EcoRI sites (pPD95.79_plag-2::Venus).
transgenes-gon-1(sig_GON) in DTCs and gon-1(FL) in musclewith transgenic animals harboring only the gon-1(sig_GON) in DTCs or with transgenic animals harboring gon-1(FL) in muscle. Gonad proliferation recovered to a greater extent in transgenic animals harboring both transgenes-gon-1(sig_GON) in DTCs and gon-1(FL) in muscle-than in transgenic animals harboring only the gon-1(sig_GON) in DTCs or only the gon-1(FL) in muscle. Our findings strongly suggest that the GON-1 protein functions both inside and outside DTCs. The GON domain functions in the ER for protein transport from the ER to the Golgi, and at the same time, the protease domain of GON-1 functions outside DTCs to digest ECM proteins. gon-1(sig_GON)::KDEL, which has an ERretention signal, rescued DTC function better than did gon-1(sig_ GON) in the gon-1 mutant (Figures 2 and 3). These data support the conclusion that the GON domain acts in the ER.
DTCs are specialized for the stimulation and navigation of gonad proliferation and generate some of the secretory or membrane proteins that are required for gonad navigation, such as UNC-5 (Su et al., 2000), and gonad proliferation, such as LAG-2 (Gao and Kimble, 1995). GPI-anchored proteins are also suggested to be involved in gonad proliferation (Murata et al., 2012). Our data imply that depletion of GON-1 impairs the transport of LAG-2, other germ cell growth factors, and DTC navigation factors to the plasma membrane. For this reason, the gonad of the gon-1 mutant fails to proliferate and undergo morphogenesis. Future studies to examine the candidate proteins transported by the GON domain function to control gonad proliferation will provide important data.
To elucidate the role of the GON domain, we performed chromophore-assisted light inactivation using the phototoxic fluorescent protein KillerRed and inactivated the GON domain. We observed the time course of the changes in ER morphology and protein secretion inhibition. In the experiments, secretory proteins accumulated in the ER within the early hours after GON-domain depletion. Cells expressing higher levels of membrane proteins contain a number of nonbranching structures, including whorls and open loops (Snapp et al., 2003). Furthermore, the accumulation of VSVG-GFP was observed in both the spherically aggregated, abnormal ER and the mesh-like, normal ER structures in the ADAMTS9 siRNA-treated cells after 20 min at the permissive temperature. These results suggest that the abnormal ER structure might be induced secondarily by the accumulation of proteins in the ER.
In human cells, siRNA-mediated knockdown of ADAMTS9 inhibited transport of the membrane protein VSVG-GFP from the ER to the Golgi and secretion of the secretory proteins PRL-GFP and AAT. Bright oval structures were observed in the ER under the same conditions. Inhibition of protein transport from the ER to the Golgi was recovered by GON-domain expression in the ER. These findings suggest that the GON domain has a common intracellular function: the export of a broader variety of cargo proteins from the ER to the Golgi in human and C. elegans.
We searched the Pfam database for proteins from other species that have the GON domain and investigated the size of those proteins, the position of the GON domain, the existence of the signal sequence, TM regions, and the metalloprotease domain. Various species have proteins with the GON domain. The size of those proteins varies from 219 to 2289 residues. Most of them have a signal sequence or a TM region, consistent with the notion that the GON domain is involved in ER-to-Golgi transport and is associated with proteins in the secretory pathway. Those proteins, however, do not necessarily have a metalloprotease or a thrombospondin domain. The metalloprotease domain is not required for GON-domain function, based on the phylogenic origins described earlier, as well as and gon-1(Sig_Pro)-RV1 for gon-1(base pairs 1-687) and gon-1(GON)-FW2 and gon-1-RV2 for gon-1(base pairs 5770-6498) as primers and pPD95.79_plag-2::gon-1 as a template. These fragments were fused together by sewing PCR using gon-1-FW2 and gon-1-RV2 as primers, and the fused fragment was inserted into the plasmid pPD95.79_plag-2 using the BamHI site.
To construct plag-2::YFP-GPI used in Supplemental Figure S5, YFP-GPI was amplified by PCR using YFP-GPI-FW and YFP-GPI-RV as primers and inserted into pPD95.79_plag-2 using the In-Fusion PCR Cloning Kit. YFP-GPI was a gift from Tom A. Rapoport (Harvard Medical School).
To construct pmyo-3::gon-1, the cDNA of gon-1 was prepared by RT-PCR using the primers of gon-1-FW and gon-1-RV and inserted into pPD114.95 (a gift from A. Fire) using the BamHI site.
To construct plag-2::ssVenus used in Figure 1, a fragment coding for SEL-1-derived signal peptide (ss) was amplified by PCR using the primers of SEL-1-ss-FW and SEL-1-ss-RV. The PCR fragment was inserted into plag-2::Venus using the In-Fusion PCR Cloning Kit (Clontech, Mountain View, CA).
To construct plag-2::Venus::SP12 used in Figure 3, the two separate fragments of SP12 cDNA and Venus were amplified by PCR using SP12-FW and SP12-RV for SP12 and Venus-FW and Venus-RV for Venus as primers. These fragments were fused together by sewing PCR using Venus-FW and SP12-RV as primers, and the fused fragment was inserted into the plasmid pPD95.79_plag-2 using the BamHI site.
To construct plag-2::gon-1(sig_Pro_GON) used in Figures 2 and  3, the two separate fragments of gon-1(base pairs 1-687) and gon-1(base pairs 5770-6498) were amplified by PCR using gon-1-FW2 of AAT-FW and AAT-RV. To construct pCMV_NHK, the AAT cDNA was amplified using NHK-FW and NHK-RV as primers and pCMV_ AAT as a template and fused with the In-Fusion PCR Cloning Kit. Aliquots of 200 ng of plasmid were transfected into the cultured cells in 35-mm dishes with Lipofectamine 2000 and analyzed at 24 h after transfection.
pCMV_AAT-Flag was generated by inserting the cDNA of AAT into pCMV_Flag, which was generated by replacing the AcGFP tag of pAcGFP with the Flag tag. To construct pCMV_AAT-Flag, the AAT cDNA was amplified using AAT-Flag-FW and AAT-Flag-RV as primers. Seventy-two hours after the ADAMTS9 or control siRNA transfection, the cells were transfected with pCMV_AAT. Aliquots of 200 ng of plasmid were transfected into cultured cells in 35-mm dishes with Lipofectamine 2000 and analyzed at 24 h after transfection.
pCMV_PRL-GFP was generated by inserting the signal sequence of PRL into pAcGFP (Voigt et al., 1996). Aliquots of 200 ng of plasmid were transfected into cultured cells in 35-mm dishes with Lipofectamine 2000 and analyzed at 24 h after transfection.

Visualizing the endoplasmic reticulum
To stain the HEK293 cells with an ER-Tracker, the cells were cultured with 100 nM ER-Tracker Blue-White DPX (Invitrogen) in the culture media for 30 min at 37°C with 5% CO 2 . After washing with phosphate-buffered saline (PBS), the cells were observed by a microscope with a 40× water-immersion objective. Images were taken with a BX-51 microscope (Olympus) with an epiluminescence light source.

VSVG trafficking
To analyze the protein transport from the ER to the Golgi, with or without the GON-domain function, 1.6 μg of VSVG-GFP (a gift from J. Lippincott-Schwartz, National Institutes of Health) and 0.1 μg of pCMV_gon-1(sig_GON) were transfected into the cells cultured in two-well chamber slides (Lab-Tek) with Lipofectamine 2000.
Cells were transfected with VSVG-GFP ts045 (a temperaturesensitive mutant of the VSVG protein) and incubated at 40°C for 16 h. To follow VSVG transport through the secretory pathway, the cells were transferred to 32°C, and after 20 min they were fixed with 4% paraformaldehyde and imaged using fluorescence microscopy.

Transmission electron microscopy
Transmission electron microscopy (TEM) analyses of C. elegans were performed as previously described (Maruyama et al., 2005). In brief, the worms were anesthetized in M9 buffer with 8% ethanol for 5 min and then cut into two or three pieces in the primary fixative solution (2% glutaraldehyde, 1% paraformaldehyde, 50 mM sodium cacodylate, 200 mM sucrose, 1 mM MgCl 2 , pH 7.4). After primary fixation for 2 h at room temperature, the pieces were postfixed in the primary fixative solution with 1% osmium tetroxide and incubated on ice for 2 h. They were then stained en bloc with 2% uranyl acetate for 2 h and dehydrated in a graded ethanol series. After replacement of ethanol with methyl methacrylate, samples were embedded in Rigolac mixture (Tamura and Kushida, 1960). Thin sections were stained with uranyl acetate and lead citrate and subjected to electron microscopic observations using one-slot copper grids precoated with Formvar. The prepared samples were observed with an H-7000 transmission electron microscope (Hitachi, Tokyo, Japan) at 75 keV.
TEM analyses of HEK293 cells were performed at the Hanaichi Ultrastructure Research Institute (Okazaki, Japan). Control siRNAtreated cells or ADAMTS9 siRNA-treated cells were fixed with 2% glutaraldehyde in 100 mM phosphate buffer, postfixed in osmium tetroxide at 4°C, dehydrated, and embedded in EPON812.
Microinjection of the described DNA constructs was performed as previously reported (Mello et al., 1991) with appropriate selection markers. Images were taken with a BX-51 microscope (Olympus, Center Valley, PA) or a confocal laser-scanning microscope LSM5 Pascal (Carl Zeiss, Jena, Germany).

RNA interference in C. elegans
The feeding RNA interference was performed as previously described (Timmons et al., 2001). Briefly, the E. coli strains of HT115(DE3) harboring double-strand RNA expression plasmids were plated on NGM medium containing 1 mM isopropyl-β-dthiogalactoside and 100 μM ampicillin. At 12 h after plating the bacteria, L1 or L4 larvae were placed on the plates and cultured at 20°C, except for the experiment shown in Supplemental Figure S6C. In Supplemental Figure S6C, the animals were cultured at 12°C to reduce the constitutive activation of xbp-1.

RT-PCR analysis
Total RNAs from the worms and HEK293 cells were extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA). Total RNA (5 mg) was used for reverse transcription with the Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) using the oligo-dT primer and amplified with the ExTaq polymerase (TaKaRa, Otsu, Japan) using the primers of sp-xbp-1-FW and sp-xbp-1-RV for the nematode xbp-1, sp-XBP1-FW and sp-XBP1-RV for the human XBP1, GAPDH-FW and GAPDH-RV for the human glyceraldehyde-3-phosphate dehydrogenase, and ama-1-FW and ama-1-RV for the nematode ama-1 (Supplemental Table S5). PCR products were separated on 10% polyacrylamide gels, stained with 5 μg/ml ethidium bromide, and photographed.
Transfection of plasmids and siRNA into HEK293 cells HEK293 cells were cultured in DMEM media supplemented with 10% fetal bovine serum at 37°C in a humidified 5% CO 2 incubator. Analyses of siRNA-mediated knockdown were performed with ON-TARGETplus SMARTpool l-005779-00 or ON-TARGETplus siCON-TROL Non-targeting siRNA (Dharmacon RNAi Technologies, Lafayette, CO) for knockdown of the ADAMTS9 gene or the nontargeting control, respectively. The cells were then transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen) and analyzed at 72 h after the transfection.
pCMV_AAT and pCMV_NHK were generated by insertion of the cDNA of AAT or NHK into a plasmid pAcGFP from which AcGCF had been removed (Clontech). AAT and NHK were gifts from Randal J. Kaufman (Sanford-Burnham Medical Research Institute). To construct pCMV_AAT, the AAT cDNA was amplified using the primers then added to the supernatants and incubated for 4 h in a cold room, washed with TBS buffer, and resuspended in the sample buffer for SDS-PAGE.

Endo H treatment
Cell extracts were denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 min. After adding 1× G5 Reaction Buffer, 500 U of Endo H (New England BioLabs, Ipswich, MA) was added, and the reaction mixture was incubated for 1 h at 37°C. Separation of reaction products was visualized by SDS-PAGE and Western blotting as described.

Chromophore-assisted light inactivation
For photoinactivation experiments, gon-1(tm3146) mutants expressing plag-2::gon-1(sig_KillerRed_GON) and plag-2::C31E10.7::Venus, plag-2::ssVenus or plag-2::xbp-1(D81)::Venus were exposed to a light-emitting diode array (572 nm) for 0, 2, 5, 10, and 24 h after the L4 stage. Transgenic animals were observed on the slide at the indicated time points, and then they were recovered from the slide to NGM plates at 20°C. The time course of the morphological changes of the ER was observed using the transgenic animals that expressed plag-2::C31E10.7::Venus. We defined an abnormal ER morphology as one that was observed as a bright fluorescent circular structure with a diameter of 1 μm or larger (Figure 4, B and C). Time courses of the accumulation of secretory proteins were observed using the transgenic animals that expressed plag-2::ssVenus. We used an oilimmersion objective with a 100× magnification and recorded the images with a DP30 charge-coupled device camera (Olympus) using a 500-ms exposure time in all of the experiments. We measured the fluorescence intensity using the ImageJ (National Institutes of Health, Bethesda, MA; Figure 4, D and E). The time course of the xbp-1 splicing was observed using the transgenic animals that expressed plag-2::xbp-1(D81)::Venus. We used an oil-immersion objective with a 100× magnification and recorded images using a DP30 camera with a 500-ms exposure time. We measured the fluorescence intensity using ImageJ (Supplemental Figure S9). Ultrathin sections were analyzed using a transmission electron microscope (JEM-1200EX; JOEL, Peabody, MA) at 80 keV.
Immunostaining HEK293 cells were plated on chamber slides ∼24 h prior to transfection. Twenty-four hours after transfection, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% Triton X-100 for 2 min. The cells were then blocked with PBS containing 5% bovine serum albumin (BSA). The cells were incubated with primary antibodies for 1 h, washed, incubated with the secondary antibodies conjugated with Alexa 488 or 594 for 1 h, washed, and observed. Images were taken with a BX-51 microscope (Olympus) and a confocal laser-scanning microscope LSM5 Pascal (Carl Zeiss). Antibodies were purchased from the following companies: anti-GM130 (BD Biosciences, San Diego, CA), anti-calnexin (BD Biosciences), anti-KEDL (Enzo Life Sciences, Plymouth, PA), and anti-AAT (Dako, Glostrup, Denmark). Secondary antibodies for immunostaining were obtained from Invitrogen.
Immunostaining of DTC was performed as previously described (Maruyama et al., 2005). In brief, transgenic animals that expressed plag-2::gon-1(sig_Venus_GON) were placed on a subbed slide in 5.0 ml of M9 containing 1 mM levamisole. Heads or tails were cut off using a 25-gauge needle, extruding the gonad and intestine. Cut animals were fixed with 1.0% paraformaldehyde in PBS for 10 min and permeabilized with PBS containing 0.1% Triton X-100 for 3 min. Samples were blocked in PBS containing 2.5% BSA for at least 30 min. Fixed worms were incubated with an anti-HDEL antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Samples were washed three times with PBS containing 0.5% BSA, incubated with a secondary antibody conjugated with Alexa 594 for 1 h, washed, and observed. Images were taken with a confocal laserscanning microscope LSM5 Pascal (Carl Zeiss).

Cycloheximide chase experiment
For analysis of AAT(NHK) degradation, HEK293 cells were transfected with pCMV-NHK. After 24 h, transfected HEK293 cells were treated with 10 μg/ml cycloheximide for 0, 1, and 2 h. Protein degradation was examined by Western blotting with an anti-AAT antibody described earlier. The amounts of the NHK protein were normalized by the α-tubulin expression as an internal control.

Immunoprecipitation
Immunoprecipitation was performed by adding protein A Sepharose beads (GE Healthcare, Piscataway, NJ) to the cell extracts, incubating for 1 h in a cold room, and washing with Tris-buffered saline (TBS; 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) buffer. After precipitation of protein A Sepharose beads, the supernatants were collected in other tubes. Anti-flag M2-agarose beads (Sigma-Aldrich) were