SURO-2/TMEM39 Facilitates Collagen Secretion through the Formation of Large COPII Vesicles

doi:10.21203/rs.3.rs-35924/v1

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SURO-2/TMEM39 Facilitates Collagen Secretion through the Formation of Large COPII Vesicles

https://doi.org/10.21203/rs.3.rs-35924/v1

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Fibrosis of various tissues is a typical disease caused by excessive production and secretion of extracellular matrix. We used Caenorhabditis elegans to investigate the formation of large transport vesicles to understand collagen secretion, a critical factor in fibrosis formation. The suro-2 mutant displays obvious defects in collagen secretion and cuticle structure including a rupture phenotype in early adults. Transmission electron microscopy exhibited that the cuticle thickness of the suro-2 mutant was severely reduced. SURO-2/TMEM39 has 8 transmembrane domains and localizes in the endoplasmic reticulum (ER) membrane. SURO-2 interacts directly with NPP-20/Sec13, a component of the coat protein II (COPII) complex responsible for ER-to-Golgi transport. SURO-2 and NPP-20 localized at the same large puncta, a large COPII vesicle enough to accommodate collagens. We report here that SURO-2/TMEM39 is highly conserved among animal species and is a specialized regulator of bulky collagen secretion rather than general transport in C. elegans.

General Cell Biology & Physiology

Epigenetics & Genomics

Stem Cell & Developmental Cell Biology

Collagen

COPII

Fibrosis

SURO-2

TMEM39

Extracellular matrix (ECM) plays an essential role in animal physiology, including cell adhesion and migration and organ formation, but also in pathological conditions, such as connective tissue disorders, autoimmune diseases, muscular dystrophy, and infectious diseases ^{1, 2}. ECM is composed of approximately 300 proteins, including fibrous proteins and proteoglycans ^{2, 3}, collagens are most abundant proteins of ECM. Collagens constitute up to 90% of ECM proteins and 30% of total proteins in the human body ^{4, 5}. In humans, 28 types of collagens have been identified, and more than 1300 mutations in collagen-associated genes are linked with genetic diseases ^{4, 6}. As with the ECM deficiency problem, excessive secretion and accumulation of ECM causes serious fibrotic diseases such as liver, kidney, heart and lung fibrosis ^{7, 8, 9}. Fibrosis that forms in solid cancer tissue is necessary or accelerated for cancer growth and metastasis ⁵. In this regard, it is important to understand the regulation and secretion of collagen and other ECM proteins.

Biosynthesis and secretion of collagen proteins is a complex process with multiple extracellular and intracellular stages. Collagen biosynthesis includes transcription and post-translational regulation, translation regulation, post-translational modification, and extracellular crosslinking. The most common and primitive collagen has a long, uninterrupted collagen repeat of typical 1,000 amino acids and consists of many Gly-X-Y tripeptide repeats where X and Y are often proline and hydroxyproline ¹⁰.

Current research on collagen secretion focuses on the early secretory pathway from the endoplasmic reticulum (ER) to Golgi apparatus. In general, secreted ER cargo is exported through coat protein II complex (COPII) vesicles. Because collagen consists of rigid triple helices up to 400 nm in length, typical COPII vesicles with a diameter of 60–90 nm are not sufficient for collagen secretion ¹¹. In the yeast Saccharomyces cerevisiae, individual genes encode inner coatomer of COPII (Sec23-Sec24 heterodimer) and outer coatomer (Sec13-Sec31 heterotetramer). However, the vertebrate genome contains several copies of COPII genes except for Sec13 ¹². Collagen transport is known to rely on several gene products including Sec23A ^{13, 14}, Sec24D ^{15, 16}, and Sec13 ¹⁷. Collagen secretion is also affected by the depletion of Sar1b, a small GTP-binding protein that initiates COPII coat assembly in the ER membrane ^{12, 18, 19}. The Sar1 cycle regulator Sedlin ²⁰, and the Sec16-interacting protein TFG1 also contribute to collagen secretion ²¹.

Several studies have demonstrated potential mechanisms for the formation of large transport vesicles, including collagen packaging and COPII vesicle size increases. The single transmembrane domain protein TANGO1 plays an important role in procollagen VII transport and formation of large vesicles ²². TANGO1 and cTAGE delay COPII vesicle formation until large enough to accommodate collagen packaging through direct binding of procollagen VII at the ER lumen and interaction with the Sec23-Sec24 heterodimer in the cytoplasm ^{22, 23, 24, 25}. Sec31 ubiquitination by the E3 ligase Cullin 3 and its specific adapter protein KLHL12 contributes to an increase in COPII coat size ²⁶. Recruitment and interaction of these proteins with COPII-related vesicles may be sufficient for the export of bulky collagen from ER ¹¹. A recent study reported that TMEM131, ranging from nematodes to humans, commonly plays an important role in regulating collagen secretion ²⁷. However, the understanding of the underlying mechanism of collagen secretion through evolutionarily large vesicles is still limited. For instance, simple animals like Caenorhabditis elegans do not have the TANGO1 homologue.

There are two main types of collagen in C. elegans: cuticle and basement collagen. Basement collagen contains two types of IV and one XVIII collagen, but cuticle collagen contains more than 170 cuticle collagen genes that support the rapid molting process associated with the synthesis and secretion of new cuticles at each stage of development ^{28, 29}. C. elegans cuticle mutants exhibit a broad range of phenotypes such as the lethal (Let), abnormal embryogenesis (Emb), dumpy (Dpy), long (Lon), blister (Bli), squat (Sqt) and roller (Rol) phenotypes ^{28, 29}. Genetic analysis of these cuticle mutants has provided valuable information regarding collagen function and ECM organization. We performed a suppressor screen using a transgenic strain of C. elegans with the Rol phenotype to identify genes involved in cuticle formation, processing, and secretion. In this study, we characterized suro–2 (suppressor-of-rolling–2) mutants in C. elegans and determined a cooperative role of SURO–2/TMEM39 and COPII in the formation of large ER vesicles essential for collagen secretion and cuticle formation.

The suro–2 mutant has serious defects in collagen secretion and cuticle structure

The suro–2 mutant was isolated from a suppressor screen of C. elegans with the Rol phenotype. The suro–2 mutation completely inhibits the Rol phenotype of the jgIs4 expressing mutant ROL–6collagen derived from the rol–6 (su1006) mutant^{30, 31, 32}. The suro–2 mutant is a small and sick Dpy, and suro–2 jgIs4 exhibits a severe Dpy phenotype compared to a single suro–2 mutant (Fig. 1a).

Another interesting suro–2 mutant phenotype is young adult lethality. Wild-type adults begin to die after laying eggs, usually 6–7 days of adult life, but retain their body shape. However, some suro–2 mutants were ruptured (Fig. 1b) and approximately 18% of suro–2 mutants died between 1–3 days (Fig. 1c). Since this burst phenotype may be related to cuticle abnormalities or disorders, we used electron microscopy to observe the cuticle structure in the suro–2 mutant. The cuticle thickness of the adult suro–2 mutant was reduced to nearly one third of the wild type (Fig. 1d). The average cuticle thickness was 604 nm in the wild type and decreased to 233 nm in the suro–2 mutant. These results suggest that SURO–2 is required for proper collagen secretion and normal cuticle formation in C. elegans.

We further examined the effect of SURO–2 on the cuticle structure using two collagen markers, ROL–6::GFP ³³ and COL–19::GFP ³⁴. These fusion proteins exhibit a striped pattern in the wild-type cuticle. Collagen-GFP expression was significantly reduced in the suro–2 mutant and the wild-type stripe pattern disappeared from the cuticle (Fig. 1e). Therefore, SURO–2 is required for the secretion of collagen, and a rupture phenotype of the suro–2 mutant may occur due to insufficient collagen supply.

SURO–2 is a homologue of mammalian transmembrane protein 39

We isolated two suro–2 alleles, jg35 and jg92. Sequencing of the D1007.5 using suro–2 (jg35) genomic DNA revealed a point mutation that resulted in the substitution of amino acid 187 with a stop codon (W187*). The wild-type suro–2 gene putatively encodes 477 amino acids (Supplementary fig. 1), while the suro–2 (jg35) mutant encodes a shorter protein composed of only 186 amino acids whose suro–2 mRNA may be degraded by nonsense RNA-mediated decay.

The suro–2 (jg92) mutantdisplays a mild Dpy phenotype compared with suro–2 (jg35). Genomic DNA sequencing of suro–2 (jg92) revealed an A>T mutation located in the noncoding region between rps–10 and suro–2 (Fig. 2a). Because D1007.4, suro–2, and rps–10 are putatively transcribed as a single operon, we compared the mRNA expression level of these genes using RT-PCR. We found that suro–2 mRNA expression decreased significantly compared to the other two genes (Supplementary fig. 1e). Because knockdown of each gene using RNAi resulted in different phenotypes (Supplementary fig. 1b) and suro–2 (jg92) showed a mild phenotype compared with the putative null suro–2 (jg35) mutant, the suro–2 (jg92) mutation and associated reduction of suro–2 mRNA expression are reliable. We speculate that jg92 allele has a problem in trans-splicing after transcription. In fact, 70% of C. elegans genes are trans-spliced after transcription using SL1 or SL2 leader sequences ³⁵. Although recent studies revealed that conserved sequences are required for trans-splicing ^{36, 37} in C. elegans, we could not link the suro–2 (jg92) mutation to any of these sequences.

SURO–2 is a homologue of human transmembrane protein 39 (TMEM39) and contains eight transmembrane (TM) domains predicted by SMART (smart.embl-heidelberg.de) and Phobius (phobius.sbc.su.se). The C. elegans genome includes one TMEM39 gene, but higher organisms have two TMEM39 genes, TMEM39A and TMEM39B. The sequence of SURO–2 shares 27.5% and 25.9% identities with human TMEM39A and TMEM39B, respectively. The C. elegans suro–2 gene expresses two isoforms resulting from alternative splicing, SURO–2A and SURO–2B (www.wormbase.org), which include 8 and 6 TM domains, respectively (Supplementary fig. 1f).

SURO–2 is a putative ER membrane protein

We generated GFP reporter constructs using the suro–2 promoter to examine the suro–2 expression pattern. Because the transgenic strain with a proximal upstream of suro–2 coding region did not express GFP, we generated another transgenic strain with a distal promoter upstream of the rps–10 coding region, and observed GFP expression in many tissues, such as the pharynx, hypodermis, body wall muscles, vulva, spermatheca, several neurons, and the intestine (Fig. 2b). We generated several GFP::SURO–2 fusion constructs using the Y37A1B.5 promoter (Y37A1B.5p) that enables to express gene products in the hypodermis ³⁸. Because we could not detect GFP expression from transgenic strains with terminal SURO–2::GFP fusions, we generated three additional GFP::SURO–2 constructs in which GFP was inserted between the cytoplasmic domains of SURO–2. The pJG720 plasmid displayed the best GFP expression, which showed fluorescence around the nuclei and relatively specific fluorescence in the cytoplasm (Supplementary fig. 2A)

We also generated several organelle markers by expressing GFP fusion proteins in the hypodermis to determine the exact subcellular localization of SURO–2. TRAM–1::GFP was used for the ER marker, and AMAN–2::GFP for the Golgi apparatus marker. TRAM–1 is the C. elegans orthologue of the translocating chain-associated membrane protein 1 ³⁹, and AMAN–2 is an alpha-mannosidase II which functions at the Golgi apparatus ⁴⁰. The pJG735 plasmid is a tdTomato version of pJG720 and expresses tdTomato::SURO–2 in the hypodermis. Most tdTomato::SURO–2 proteins overlapped exactly with TRAM–1::GFP, but not with AMAN–2::GFP puncta (Fig. 2c). Because other ER markers such as GFP::KDEL and GFP::PISY–1 also overlapped with tdTomato::SURO–2 (Supplementary fig. 2B), we concluded that SURO–2 mainly localized to the ER membrane.

Because suro–2 encodes two isoforms via alternative splicing, we examined the subcellular localization and functional differences of these two proteins. To examine the expression of SURO–2A and SURO–2B in the hypodermis, we generated a transgenic strain expressing tdTomato::SURO–2A and GFP::SURO–2B simultaneously. We found that these two proteins overlapped in most area (Fig. 2d), suggesting SURO–2A and SURO–2B function at the same ER membrane. Next, we performed rescue experiments using the suro–2 jgIs4 strain to compare protein function involved in cuticle formation. We crossed suro–2 jgIs4 with transgenic strains expressing GFP::SURO–2A or GFP::SURO–2B. The suro–2 jgIs4 strain expressing the GFP::SURO–2A transgenic product recovered the Rol phenotype, whereas the suro–2 jgIs4 strain expressing GFP::SURO–2B did not (Fig. 2e). Therefore, hypodermal SURO–2A expression is required and sufficient for proper cuticle formation.

SURO–2 is closely related to COPII in relation to collagen secretion

A large-scale yeast two-hybrid screen for protein networks and a genome-wide prediction of C. elegans genetic interactions proposed associations between D1007.5 (SURO–2) and nucleopore protein–20 (NPP–20) ^{41, 42}, the C. elegans orthologue of human protein Sec13. Sec13 is a component of the COPII complex originally discovered in S. cerevisiae and participates in the early collagen secretory pathway ^{43, 44}. Another role of Sec13 relates to its function as a nucleopore protein during nuclear envelope reassembly ⁴⁵ and is associated with the GATOR2 complex that regulates the mTORC1 complex ⁴⁶. Amidst varied functions, we focused on its potential role in the collagen secretory pathway to understand the relationship between SURO–2 and NPP–20, because SURO–2 is localized at the ER and participates in cuticle formation. We examined the knockdown phenotype of npp–20 via RNAi to compare its phenotype with that of the suro–2 mutant. Like suro–2 mutants, npp–20 RNAi suppressed the Rol phenotype of jgIs4 (Fig. 3a), implying a close relationship between suro–2 and npp–20. Therefore, we performed RNAi-mediated knockdowns of other COPII components to examine their functions in cuticle formation. Knockdown of most COPII genes resulted in Rol suppression. Knockdown of sar–1 and tfg–1 also resulted in Rol suppression (Fig. 3b). SAR–1 is the orthologue of human Sar1 GTPase which is the key regulator of COPII vesicle formation ¹⁹. TFG–1 associates with SEC–16 and also functions in early collagen secretion ⁴⁷. Collectively, these results suggest that SURO–2 and COPII may function in the same secretory pathway.

Following Rol suppression by COPII knockdown, we determined collagen secretion was affected by COPII RNAi. We compared COL–19::GFP expression in the cytoplasm to identify potential deficiencies in collagen secretion when SURO–2 or NPP–20 was depleted by RNAi. COL–19::GFP was evenly distributed in the wild-type background, but COL–19::GFP accumulated in the cytoplasm by SURO–2 and NPP–20 knockdowns (Fig. 3c). We observed larger COL–19::GFP puncta in SURO–2-depleted worms than in NPP–20-depleted worms, implying that SURO–2 is more specialized in collagen secretion than NPP–20. Other components of COPII were depleted by RNAi and also resulted in COL–19::GFP accumulation in the cytoplasm (Fig. 3d). COPII plays a major role in early secretion of collagens from ER-to-Golgi apparatus ^{17, 23}, and our experimental data supported this role as described in previous studies. Therefore, SURO–2 and COPII are closely related with respect to collagen transport and cuticle formation.

We validated the expression of COPII proteins if they colocalize in C. elegans. We generated transgenic strains expressing NPP–20::GFP and SEC–24.1::tdTomato simultaneously. Many NPP–20::GFP and SEC–24.1::tdTomato proteins were punctate and overlapped in the cytoplasm, whereas NPP–20::GFP also expresses in the nucleus (Fig. 3e).

SURO–2 interacts directly and is located together with NPP–20

SURO–2 and COPII are collectively involved in collagen secretion, and hypodermal expression is important for cuticle formation. We performed a glutathione S-transferase (GST) pulldown analysis to examine the direct interaction of SURO–2 and NPP–20. Because we were unable to express a SURO–2 protein including its TM domains in E. coli, we used its two long loop domains exposed to cytoplasm (Fig 4A). We found that NPP–20 and SURO–2 interacted directly through the 6th loop (L6) of SURO–2 using in vitro binding analysis (Fig. 4b).

We constructed 11 plasmids expressing series of YFP::SURO–2 deletion proteins to examine in vivo expression and function of SURO–2 domains (Supplementary fig. 3A). Most YFP::SURO–2 proteins exhibited similar expression patterns to intact protein excepting SURO–2C3, which has only C-terminus of SURO–2 and expressed like simple GFP expression in the cytoplasm and nucleus, while some proteins yielded strong punctate fluorescence. In particular, YFP::SURO–2 proteins having the L6 domain with the C-terminus formed large bright puncta (Supplementary fig. 3B). These results correlate with in vitro interactions of SURO–2 and NPP–20 and suggest the importance of the L6 domain of SURO–2 for in vivo complex formation.

We used the jgIs47 strain which expresses tdTomato::SURO–2 and NPP–20::GFP simultaneously to examine in vivo colocalization of SURO–2 and NPP–20. In most cases, tdTomato::SURO–2 localized at the ER membrane as indicated by comparison of ER markers (Fig. 2b). Occasionally, the tdTomato::SURO–2 expressing strain displayed large fluorescent puncta, and their ER membrane-associated expression was relatively weak in the same cell. NPP–20::GFP appeared as variable-sized puncta. When tdTomato::SURO–2 proteins formed large fluorescent puncta, large NPP–20::GFP puncta overlapped with tdTomato::SURO–2. When tdTomato::SURO–2 spread throughout the ER membrane, NPP–20::GFP proteins were dispersed as smaller puncta (Fig. 4c). Similarly, SURO–2::GFP and SEC–24.1::tdTomato colocalized as large puncta, and small SEC–24.1::tdTomato puncta exist solitarily (Fig. 4d). To further clarify the relationship between SURO–2 and NPP–20, fluorescence was quantified by observing at high resolution using a confocal microscope. When NPP–20::GFP independently formed small puncta, the intensity of NPP–2::GFP and tdTomato::SURO–2 was low and fluorescence peaks did not overlap (Fig. 4e left panel). When NPP–20::GFP formed large puncta with tdTomato::SURO–2, the intensity of the two fluorescence was high and peaks overlapped (Fig. 4e right panel).

Super resolution images using Airyscan determined the identity of the puncta formed by NPP–20::GFP and tdTomato::SURO–2. The processed images of both small and large puncta appeared as circular forms. As a result, each punctum can be assumed to be a fluorescence image of the vesicle. The small puncta composed of NPP–20::GFP was around 100 nm in diameter (Fig. 4f left panel). Whereas, large puncta of NPP–20::GFP and tdTomato::SURO–2 were often larger than 400 nm in diameter (Fig. 4f right panel). This result implies that SURO–2 is required and facilitates large COPII vesicle formation.

Large COPII vesicles were actively produced during molting

We observed fluorescence over time from mid L4 larvae to investigate when large vesicles formed. In the mid L4 stage, tdTomato::SURO–2 expressed at ER membrane and NPP–20::GFP formed small puncta. From the late L4 stage, large puncta composed of tdTomato::SURO–2 and NPP–20::GFP began to form, and increased until young adults. The peak time of large vesicle formation represented by NPP–20::GFP puncta size and intensity is around 24 hours from the mid L4 stage. Passing the gravid adult stage, tdTomato::SURO–2 expression disappeared in the hypodermis and NPP–20::GFP expressed as small puncta (Fig. 5a). This observation implies that large vesicle formation is highest when new cuticles form, and most SURO–2 proteins in the hypodermis are consumed for making large vesicle in contrast to NPP–20 which existed as small vesicles in late adults.

Following strong interaction of SURO–2 and NPP–20, we examine the relationship of these proteins by depleting SURO–2 or NPP–20 using RNAi. SURO–2 RNAi resulted in reduction of NPP–20::GFP expression, both of intensity and large puncta formation in compared to control RNAi. When NPP–20 was depleted by RNAi, tdTomato::SURO–2 disappeared completely (Fig. 5b). However, small NPP–20::GFP puncta existed after SURO–2 depletion. These results suggest that SURO–2 is specialized for large vesicles rather than general COPII vesicles. The mutual stabilization and cooperation of SURO–2 and NPP–20 proteins seem to form large vesicles to facilitate collagen transport from the ER.

We also investigated if COPII regulators are required for large vesicle formation as their knockdown suppressed the Rol phenotype (Fig. 3b). We performed sar–1 RNAi using the jgIs56 strain expressing tdTomato::SURO–2 and NPP–20::GFP. To observe young adults, we transferred L4 larvae to sar–1 RNAi plates because sar–1 RNAi from early larvae resulted in larval lethality. After 24 hours from L4, sar–1 knockdown resulted in significant reduction of large vesicles compared with control RNAi. The mean vesicle size and intensity of NPP–20::GFP decreased by SAR–1 depletion (Fig. 5c). From this result, we know that SURO–2 associated large vesicle formation is basically dependent on COPII and its regulator.

Cytoplasmic long loop domains are essential for SURO–2 expression and function

The domain study and in vitro binding assay of SURO–2 implied the importance of cytoplasmic long loop domains, L4 and L6. We generated transgenic strains expressing NPP–20::GFP together with L4 deletion form of tdTomato::SURO–2A (tdTomato::SURO–2ΔL4) or L6 deletion (tdTomato::SURO–2ΔL6) to prove critical roles of L4 or L6 domains in vivo. In the wild-type background, tdTomato::SURO–2ΔL4 forms large puncta with NPP–20::GFP superior to tdTomato::SURO–2A and tdTomato::SURO–2B. In contrast to tdTomato::SURO–2ΔL4, tdTomato::SURO–2ΔL6 could not form large vesicles (Supplementary fig. 4A). This result implies that L6 is required for stable SURO–2 expression and large vesicle formation when the endogenous wild-type SURO–2 exists. We wondered that L4 domain functions as a negative role for large vesicle formation on the contrary to L6. To examine this, we mated these transgenic strains with the suro–2 (jg35) mutant. The result is that tdTomato::SURO–2ΔL4 and tdTomato::SURO–2ΔL6 rarely expressed in the suro–2 mutant background. Only tdTomato::SURO–2A expressed well and rescued suro–2, but others including tdTomato::SURO–2B didn’t rescue. Like these results, NPP–20::GFP expressed together with tdTomato::SURO–2ΔL4, tdTomato::SURO–2ΔL6 and tdTomato::SURO–2B did not form large vesicles well in the suro–2 mutant background (Fig. 6a). This result means that the previous expression data were biased by endogenous SURO–2, and both L4 and L6 are essential for SURO–2 expression and large vesicle formation.

We further investigated to find out important regions of L4 and L6 domains by sequence alignment of SURO–2 and human TMEM39A and B. The posterior region of L4 domain and the anterior region of L6 domain are highly conserved compared with other regions. Seven out of ten amino acids from 232 to 241 and eight out of eleven amino acids from 318 to 328 are positive among three proteins (Fig. 6b). We generated 10 aa deletion (tdTomato::SURO–2Δ10) or 11 aa deletion (tdTomato::SURO–2Δ11) expression constructs to examine the importance of these conserved amino acids in L4 and L6 domains. In the wild-type background, tdTomato::SURO–2Δ10 expression was stronger than tdTomato::SURO–2A similar to tdTomato::SURO–2ΔL4 expression. tdTomato::SURO–2Δ11 and tdTomato::SURO–2Δ10Δ11 did not express well similar to tdTomato::SURO–2ΔL6 (Supplementary fig. 4B). In the suro–2 mutant background, any tdTomato::SURO–2 deletion proteins did not express well. As expected, the size and intensity of NPP–20::GFP puncta when expressed together with tdTomato::SURO–2Δ10 or tdTomato::SURO–2Δ11 decreased significantly in the suro–2 mutant (Fig. 6c).

SURO–2 is required for SURO–1/Carboxypeptidase A secretion

SURO–1 is a carboxypeptidase A (CPA) and is required for normal cuticle formation. Since the suro–1 mutant exhibited a mild Dpy compared with the suro–2 mutant that is a severe small Dpy, SURO–1 is one candidate cargo protein of the SURO–2 vesicle. In particular, SURO–1::DsRed is also secreted to the cuticle as a large vesicle ³². To examine that SURO–2 is required for SURO–1 secretion, the jgIs32 integration line expressing SURO–1::DsRed was crossed with the suro–2 mutant. SURO–1::DsRed exhibited a typical cuticle pattern in wild type, but it was barely detected in the suro–2 mutant excepting marginal regions of the cuticle. Whole fluorescence intensity of SURO–1::DsRed also decreased significantly in the suro–2 mutant (Fig. 7a). Next, we generated a transgenic strain expressing SURO–1::GFP and tdTomato::SURO–2 to examine that SURO–1 is localized at the SURO–2 vesicle. Most SURO–1::GFP proteins were exactly localized at the same puncta of tdTomato::SURO–2 (Fig. 7b). These results indicate that one of the collagen-modifying enzymes, SURO–1/CPA, is secreted by large vesicles composed of SURO–2.

In conclusion, the results of this work are summarized in a simple model (Fig. 7c). Soluble proteins are transported from the ER by conventional COPII vesicles with an average diameter of 80 nm. On the other hand, many proteins involved in ECM should be transported from the ER using large vesicles. Both types of vesicles, large and small, contain COPII and commonly require regulators such as Sar1. The conserved protein SURO–2/TMEM39 is necessary for the formation of this large vesicle and may function as the basic mechanism of bulky secretion, from simple animals to mammals.

We have identified novel genes involved in collagen secretion and ECM remodeling using C. elegans to provide basic knowledges for solving fibrotic diseases. SURO–2/TMEM39, a suro–2 gene product, is a highly conserved protein found in animals. The secretion level of collagen proteins was drastically reduced in the suro–2 mutant, indicating SURO–2, rather than TANGO1/cTAGE5, may play a fundamental role in bulky collagen secretion like recently reported TMEM131 ²⁷. Since TANGO1/cTAGE5 have only been found in higher animals, the TANGO1-cTAGE system may have evolved to enable transport of diverse collagens. In fact, collagen VII, a main cargo of TANGO1-associated COPII vesicles, does not exist in C. elegans. However, we cannot dismiss the possible existence of protein other than SURO–2 that is directly involved in bulky collagen secretion because the suro–2 (jg35) mutant phenotype was less severe than that of a single essential collagen mutant. In addition, the cuticle still formed without SURO–2, indicating cuticle formation is not wholly depended on SURO–2 (Fig. 1d). Nevertheless, our data suggest that SURO–2 is a common key component for collagen secretion among diverse animal species.

Two TMEM39 genes exist in the human genome, as opposed to a single gene in C. elegans. TMEM39A seems to be closely associated with the human immune system, as several genome-wide association studies have reported relationships between TMEM39A polymorphisms and several autoimmune diseases, such as multiple sclerosis (MS) ⁴⁸ and systemic lupus erythematosus ⁴⁹. For example, the first genome-wide association study of MS patients identified KIF21B and TMEM39A as MS susceptibility loci ⁴⁸. MS, which affects the central nervous system, is likely to be linked to the collagen secretion function of TMEM39A in the fact that it is clinically indistinguishable from collagen disorders ^{50, 51}. KIF21B is a kinesin motor protein that functions in Golgi-to-ER retrograde transport mediated by COPI vesicles ⁵². Likewise, Arf1 which is a key regulator of COPI vesicle formation, seems to be required for proper cuticle formation, because knockdown of arf–1, the C. elegans Arf1 orthologue, resulted in Rol suppression (our unpublished data). Defects in either TMEM39A or KIF21B could lead to perturbation of the ER-to-Golgi or Golgi-to-ER secretion of certain proteins involved in the immune system and MS. No functional study of TMEM39B has been published to date, although one previous report indicates TMEM39B is highly expressed in diffuse large B-cell lymphomas ⁵³. Thus, our results regarding the functionality of SURO–2/TMEM39 could provide preliminary information to guide studies of TMEM39A/B and their roles in human disease.

COPII and its associated gene products are required for collagen transport from the ER in vertebrates ¹². In C. elegans, SEC–23/Sec23 was reported to function in collagen secretion during embryogenesis ⁵⁴. Our RNAi experiments showed that most COPII genes suppressed the Rol phenotype and induced collagen accumulation in the cytoplasm as observed in suro–2 knockdown. However, SURO–2 depletion exhibited more obvious phenotypes than the COPII gene knockdown in collagen secretion (Fig. 3). Because COPII is essential for the export of general proteins including soluble and membrane proteins, COPII depletion resulted in more pleiotropic and detrimental phenotypes than SURO–2 was depleted. The suro–2 (jg35) mutant, which is expected to be a null mutation, is alive, while most of the COPII gene RNAi leads to embryonic death. Thus, SURO–2 may specifically facilitate bulky secretion of collagens and collagen processing enzymes, rather than of general proteins. However, SURO–2 may be involved in secretion of other proteins besides collagens, as suro–2 was expressed throughout various cell types that do not produce collagens (Fig. 2b).

SURO–2 protein has eight putative TM domains and two long cytoplasmic loop domains. We determined that the L6 cytoplasmic domain directly binds NPP–20/Sec13. Due to its eight TM domains, SURO–2/TMEM39’s movement might be more restricted than that of NPP–20/Sec13, a soluble cytoplasmic protein. Therefore, SURO–2 may recruit Sec13-Sec31 outer coatomers through direct interaction with Sec13 when a large vesicle is forming. SURO–2 and NPP–20 localization patterns also suggest that SURO–2 recruits NPP–20 to large collagen transport vesicles. Large puncta of NPP–20::GFP localization overlapped exactly with those of tdTomato::SURO–2, although smaller puncta of NPP–20::GFP did not (Fig. 4). Secretion of SURO–1, a CPA involved in collagen processing, was also dependent on SURO–2 function (Fig. 7a). SURO–1::DsRed proteins were secreted as large puncta in the cytoplasm and initially considered to be secretory granules because of their large size ³². Taken together, SURO–1 function is interpreted as a subset of SURO–2 because SURO–1 and SURO–2 localized at the same large puncta (Fig. 7b), and the suro–1 mutant has a much weaker phenotype than the suro–2 mutant.

Cytoplasmic regions of SURO–2 are composed of 5 domains including N-terminus, L2, L4, L6 and C-terminus (Fig. 4a). We tried to clarify the function around L4 and L6 domains, because they are long enough to have roles compared with the short L2 domain. Both L4 and L6 domains are required for SURO–2 expression, and particularly, L6 domain binds NPP–20/Sec13 directly. Because large vesicles are composed of SURO–2 and COPII, NPP–20 interacting L6 domain has a critical role in collagen secretion. SURO–2 and human TMEM39 proteins have highly conserved regions in L4 and L6 domains. These conserved 10 amino acids in L4 and 11 amino acids in L6 are required for L4 and L6 function to express SURO–2 itself and form large vesicles. The greatest difference between L4 and L6 was that the L4 deletion form increased in the wild type but was not expressed well in the suro–2 mutant. When endogenous SURO–2 and L4-deficient SURO–2 coexist, there appears to be a complex interaction between SURO–2 proteins.

Because the secondary protein structure and binding partner of SURO–2 are unlike TANGO1, a single-TM protein that interacts with Sec23-Sec24 inner coatomers ^{24, 41}, mechanisms of collagen-packaging vesicle formation between the two proteins may differ. TANGO1 has a long tail and delays COPII vesicle formation by interacting with collagen VII directly ⁴¹. However, both termini of SURO–2/TMEM39 are exposed to the cytoplasm, and only four short loop regions (L1, L3, L5 and L7) composed of less than 20 amino acids are located on the luminal side of the ER (Fig. 4a). We propose that SURO–2 directly incorporates into the COPII membrane and reduces its flexibility via SURO–2’s eight TM domains and four luminal loop domains. This complex of ER membrane and SURO–2 proteins may look like sewn buttons on fabric. The inflexible domains of the COPII membrane formed by SURO–2 may inhibit the formation of standard-sized COPII vesicles and instead induce spontaneous formation of a larger COPII-SURO–2 vesicle. This hypothesis requires additional investigation using biochemical, structural, and biophysical approaches, although we propose a model for early collagen secretion based on our data presented here (Fig. 7c). In mammals, HSP47, which functions as a collagen chaperone, is present in the ER lumen ⁵⁵, and the presence of HSP47 orthologue has not been reported in nematodes. Recently reported TMEM131 recruits premature collagen monomers through the bacterial PapD chaperone-like domain and functions in collagen assembly and secretion. The C-terminal region of TMEM131 interacts with TRAPPC8, a component of the TRAPP tethering complex, and functions in collagen secretion ²⁷. In this regard, SURO–2/TMEM39 and TMEM–131/TMEM131 seem to cooperate in collagen secretion.

In summary, we elucidated novel functions of SURO–2/TMEM39 in C. elegans, thus providing valuable insights into bulky protein secretion, matrix biology, and TMEM39A-associated diseases including cancer fibrosis in higher animals.

C. elegans cultures and strains

The wild-type N2 strain and other mutant strains were fed on E. coli (OP50) growing in the standard nematode growth media (NGM) as described previously ⁵⁶. The C. elegans strains used in this study are jgIs4, jgIs5, jgIs32, jgIs47, jgIs56, and TP12 (kaIs12).. The jgIs4 strain expresses mutant ROL–6 collagen originated from rol–6 (su1006), and jgIs5 expresses ROL–6::GFP. The jgIs32 strain expresses SURO–1::DsRed, and jgIs47 expresses both tdTomato::SURO–2 and NPP–20::GFP by the Y37A1B.5 promoter. The jgIs56 strain expresses tdTomato::SURO–2 and NPP–20::GFP by the col–12 promoter. These integration lines were generated by UV irradiation and outcrossed with N2 males four times.

Mapping and cloning of suro–2 mutants

We used a SNP mapping method to identify suro–2 (jg35) mutation as previously described ⁵⁶. More than 1,000 F2 progeny that exhibited Dpy and non-Rol phenotypes were selected from the cross of suro–2 (jg35) jgIs4 and Hawiian CB4856 male. Several F3 worms from each F2 plate were lysed, and their genomic DNA was used for PCR templates. PCR products were digested by restriction enzymes specific to each SNP and analyzed by 2% agarose gel electrophoresis. The RNAi clones for 12 candidate genes in the mapped region were originated from the Ahringer library ⁵⁷. Several L4-stage jgIs4 worms were transferred to each RNAi plate, and F1 progeny were observed. The WRM067cA7 fosmid and 8 kb PCR products, including three genes in the same operon, were used for the rescue experiment. We generated transgenic worms and mated each transgenic line with suro–2 mutants because direct microinjection ruptured suro–2 mutants. Finally, PCR-amplified D1007.5 from the suro–2 mutant was sequenced to detect molecular resin (Cosmogenetech, Seoul, South Korea). Whole-genome sequencing of suro–2 (jg92) was performed by Macrogen (Seoul, South Korea).

Fluorescent protein fusion constructs and microinjection

We used GFP-encoded pPD95.77 and pPD117.01 Fire vectors and generated genes encoding additional fluorescent proteins (FP), such as YFP, DsRed, and tdTomato, via PCR for incorporation into Clontech vectors, such as pEYFP. For hypodermal expression, we used the Y37A1B.5 promoter or col–12 promoter, and pJG452 (Y37A1B.5p:::GFP) was constructed from Y37A1B.5p and pPD95.77 using HindIII and PstI. Primers used for amplification of the Y37A1B.5 promoter region were Y37A1B.5–03 (5 TAT AAG CTT AGC GAG TGA CAT TTG CCG 3) and Y37A1B.5–02 (5 TAT CTG CAG TTT GGT TTT TGG GAT TTT TGA TCT GC 3). We observed strong GFP expression in the hypodermis of the transgenic strain carrying pJG452. The final concentration of microinjection mixture was 150 µg/ml, including 75 µg/ml pRF4 (Rol) or 5 µg/ml pCFJ90 (myo–2p:::mCherry), which were used as injection markers. SURO–2-or COPII-FP fusion plasmids were injected at 20–50 µg/ml concentration, and the pBluescript SK+ plasmid was added to adjust the final DNA concentration when necessary.

Plasmids used in this study were pJG405 (suro–2p:::NLS::GFP), pJG476 (Y37A1B.5p:::GFP::SURO–2L4), pJG477 (Y37A1B.5p:::SURO–2N::GFP::SURO–2C), pJG493 (Y37A1B.5p:::NPP–20::GFP), pJG496 (Y37A1B.5p:::NPP–20::DsRed), pJG519 (Y37A1B.5p:::AMAN–2::GFP), pJG527 (Y37A1B.5p:::GFP::TRAM–1), pJG536 (Y37A1B.5p:::GFP::PISY–1), pJG601 (sec–24.1p:::NLS::GFP), pJG602 (sec–24.2p:::NLS::GFP), pJG663 (Y37A1B.5p:::GFP::KDEL), pJG683 (Y37A1B.5p:::SEC–24.1::tdTomato), pJG719 (Y37A1B.5p:::SP::GFP), pJG720 (Y37A1B.5p:::GFP::SURO–2A), pJG735 (Y37A1B.5p:::tdTomato::SURO–2A), pJG789 (Y37A1B.5p:::GFP::SURO–2B), pJG819 (Y37A1B.5p:::YFP::SURO–2), pJG1079 (Y37A1B.5p:::SURO–2N::GFP::SURO–2C), pJG1090 (Y37A1B.5p:::tdTomato::SURO–2L4), pJG1111 (Y37A1B.5p:::YFP::SURO–2L4), pJG1114 (Y37A1B.5p:::YFP::SURO–2L6). pJG1115 (Y37A1B.5p:::YFP::SURO–2(SP)), pJG1117 (suro–2p:::GFP), and pJG1124 (Y37A1B.5p:::YFP::SURO–2AC).

RNA interference

Most RNAi clones that were originated from the Ahringer library ⁵⁷ were kindly provided by Dr. J. Lee (Seoul National University). Additionally, we constructed RNAi clones for suro–2 using its cDNA and L4440 vector. The 1.4 kb suro–2 cDNA was amplified by PCR using the wild-type N2 cDNA as templates. The PCR fragments were cloned into L4440 vector using SalI and BamHI sites. Primers used for suro–2 RNAi clone were D1007.5–03 (5ATG TCG ACA TGC CGC CTC GAA GAC G 3) and D1007.5–23 (5 TGG ATC CTT ATC GTT CTT GTT GAA GTT GAT GC 3). We transferred different staged worms for each gene RNAi according to the extent of RNAi effect. L4 stage worms were transferred for control, sec–31 and suro–2 RNAi, and gravid adult worms for npp–20 and sec–16 RNAi. F1 progeny were observed in these cases. However, the effect of sec–12, sec–23, sec–24.1, sar–1 and tfg–1 RNAi was very severe, and L1-L2 larvae were transferred and observed when they grew up. The empty vector L4440 was used for control.

GST pull-down and western blot analyses

The pGEX4T1 GST vector system (GE helthcare, Wauwatosa, WI, USA) or GST-SURO–2 and the pRSET vector system (Thermo-Fisher, Waltham, MA, USA) were used for 6×His-NPP–20 protein expression in E. coli (BL21). The soluble fraction of the bacterial lysate was prepared by sonication and centrifugation. The soluble fraction containing GST or GST-SURO–2 was mixed with glutathione-agarose beads (G–4510; Sigma, St. Louis, MO, USA) and washed five times with sonication buffer (100 mM NaCl, 25 mM Tris-HCl (pH8.0), 10% glycerol). The bacterial lysate containing 6×His-NPP–20 was mixed with G-agarose beads bound to GST or GST-SURO–2 and incubated overnight at 4°C. Each G-agarose bead suspension was then washed five times with sonication buffer and boiled for 2 min after SDS-PAGE sample buffer was added. Each sample was loaded and electrophoresed on the 12% SDS-PAGE gel. Rabbit anti-NPP–20 antibodies, generated by immunizing synthetic NPP–20 peptides composed of 14 amino acids (IKVPRDNKEREGMS) in C-terminal region of NPP–20 (Peptron, Daejeon, South Korea), were used to detect 6×His-NPP–20. Anti-NPP–20 serum was purified by affinity column chromatography (SulfoLink Immobilization kit; Thermo-Fisher) according to the supplier’s instruction.

Transmission electron microscopy

The wild-type N2 and suro–2 (jg35) mutant were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (PB, pH 7.4) overnight at room temperature. The samples were then post-fixed in 1% OsO₄ and 1.5% potassium ferrocyanide for 1 h at room temperature and stained by 0.5% uranyl acetate overnight at 4°C and dehydrated in the graded ethanol series. After polymerization in Epon for 2 days at 60°C, the glass coverslips were detached from epon block. Samples were sectioned by EM UC7 (Leica, Wetzlar, Germany) ultramicrotome to 70 nm thickness and contrasted with 0.5% uranyl acetate and lead citrate. The specimens were examined in Tecnai G2 Spirit Twin transmission electron microscopy (FEI, Hillsboro, OR, US; Instrumentation was used in Korea Basic Science Institute) operated at 120 kV. Images were recorded on 4Kx4K Ultrascan 895 CCD (Gatan, Pleasanton, CA, USA).

Fluorescence microscopy and statistical analysis

Fluorescence microscopy was performed using an Axio Imager A2 compound and LSM880 confocal microscopes (Zeiss, Oberkochen, Germany) and images were processed using Axiovision Rel.4.5 software (Zeiss), Photoshop (Adobe systems, San Jose, CA, USA) and Image J (https://imagej.nih.gov/ij). Worms were prepared with M9 buffer containing 2.5 mM levamisole. Statistical analyses including t-test in this study were carried out with SigmaPlot software (Systat software, San Jose, CA, USA). P values of <0.05, <0.01, and <0.001, indicated statistical significance and are denoted with *, **, or ***, respectively.

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Acknowledgements

We appreciate Dr. Junho Lee and his laboratory members for providing RNAi clones, and Dr. Donghoon Lee for providing useful comments. Nematode strains such as TP12 were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. This work was supported in part by grants from the National Cancer Center of Korea (NCC-1610290, NCC-1810932) and the National Research Foundation (NRF-1531940). EM study was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (NRF-2015R1C1A1A01053611).

Author information

These authors contributed equally: Kiho Lee and Jee Young Sung

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Contributions

K.L., J.Y.S., Y.K., H.S.J. and J.S. designed research; K.L., J.Y.S., S.L., G.L., K.J.J, J.M.C. and J.S. performed experiments; Y.K., H.S.J. and J.S. analyzed data and wrote the paper.

Corresponding authors

Yong-Nyun Kim. Research Institute, National Cancer Center, 323 Ilsan-ro, Goyang-si, Gyeonggi-do, 10408, Korea. Tel: 82-31-920-2415. E-mail: [email protected]

Jaegal Shim. Research Institute, National Cancer Center, 323 Ilsan-ro, Goyang-si, Gyeonggi-do, 10408, Korea. Tel: 82-31-920-2262. E-mail: [email protected]

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There is NO Competing Interest.

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SURO-2/TMEM39 Facilitates Collagen Secretion through the Formation of Large COPII Vesicles

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Abstract

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Introduction

Results

The suro–2 mutant has serious defects in collagen secretion and cuticle structure

SURO–2 is a homologue of mammalian transmembrane protein 39

SURO–2 is a putative ER membrane protein

SURO–2 is closely related to COPII in relation to collagen secretion

SURO–2 interacts directly and is located together with NPP–20

Large COPII vesicles were actively produced during molting

Cytoplasmic long loop domains are essential for SURO–2 expression and function

SURO–2 is required for SURO–1/Carboxypeptidase A secretion

Discussion

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