Collagen is the major molecular component of connective tissues, and the most 21 abundant protein in animals (1) . Collagen dysregulation causes many human disorders, 22
including autoimmune diseases, brittle bone diseases (too little collagen), tissue 23 fibrosis (too much collagen) and aging-related disorders (2-7). The multi-step 24 biosynthesis of mature collagen by the cell is a complex process and involves 25 procollagen gene transcription and protein translation, posttranslational modification, 26 assembly into procollagen trimers inside the endoplasmic reticulum (ER), vesicular 27 secretion from ER, extracellular peptide cleavage and cross-linking into collagen fibers 28 (1, 8) . 29 Specific mechanisms underlying the secretion of procollagen still remain poorly 30 understood. In general, specialized intracellular vesicles defined by the coat protein 31 complex II (COPII) transport most secreted proteins, including procollagen, from the 32 ER to the Golgi apparatus (9, 10). Sec23, Sec24, Sec13 and Sec31 comprise COPII 33 coat proteins, while the transport protein particle (TRAPP) complex acts a key tethering 34 factor for COPII vesicles en route to the Golgi (11-13). Typical COPII vesicles are 60 35 to 80 nm in diameter, which is not sufficient for transporting procollagen trimers with 36 up to 300 to 400 nm in length (14). In mammals, large-size COPII-coated vesicles may 37 transport procollagen from the ER to the Golgi apparatus. TANGO1, a transmembrane 38 protein at the ER exit site, mediates formation of specialized collagen-transporting 39 vesicle and recruitment of procollagen (14-16). The N-terminal SH3-like domain of 40 TANGO1 binds to the collagen chaperone HSP47 in the ER lumen, recruiting 41 procollagens to the ER exit site (17). Its C-terminal proline-rich domain (PRD) servers 42 as a COPII receptor by interacting with the inner shell proteins Sec23/Sec24 (18). The 43 coil-coil domain of TANGO1 forms a stable complex with cTAE5 and SEC12, which is 44 particularly enriched around large COPII carriers for procollagen (19) . Through its 45 membrane helices, TANGO1 organizes ER exit sites by creating a lipid diffusion barrier 46 and an export conduit for collagen (20) . While requirement of TANGO1 for secretion 47 may depend on specific collagen types, it remains unclear whether TANGO1's 48 functions are broadly conserved in all animals (21, 22) . 49 Caenorhabditis elegans produces over 180 collagen members that constitute the 50 cuticle and basement membranes, encodes conserved homologs of COPII/TRAPP 51 proteins, yet lacks apparent TANGO1 homologs (23-26). This indicates that 52 evolutionarily conserved and TANGO1-independent mechanisms may exist in C. 53 elegans to regulate procollagen secretion. From a genome-wide RNAi screen for 54 genes affecting ER stress response, we previously identified tmem-131 that defines a 55 broadly conserved family of proteins important for procollagen assembly and secretion 56 (22). Mutations in specific collagen genes, conserved COPII/TRAPP-encoding 57 homologs, and impairment of collagen biosynthetic pathway components are known 58 to result in a range of phenotypes including ER stress response, abnormal cuticle-59 associated morphology (Blister and Dumpy), and early death or growth arrest (23). Table) and found that tmem-39 RNAi knock-down strongly reduced 114 abundance of the COL-19::GFP reporter (Fig 2A) . COL-19 is a C. elegans exoskeleton 115 collagen that is secreted by the underlying hypoderm and required for integral structure 116 of the cuticle (23). The C-terminal GFP-tagged COL-19 reporter enables highly robust 117 and tractable visualization of the cuticle morphology and to identify defects in the 118 collagen production machinery (27) Using confocal microscopy to characterize the structure of hypodermal cuticle, we 140 found that in control RNAi animals, COL-19::GFP is enriched in the hypoderm, 141 constituting regular annular furrows and lateral alae of the cuticle (Fig 2B) . In the tmem-142 39 RNAi animals, COL-19::GFP appeared to be clustered in the intracellular region of 143 hypoderm, and largely absent in the cuticle (Fig 2B) . We further analyzed the 144 abundance and composition of COL-19::GFP proteins by Western blot. Besides strong 145 reduction of overall COL-19::GFP abundance, tmem-39 RNAi markedly increased the 146 soluble "premature" monomeric procollagens, while decreased the insoluble fraction 147 of cross-linked multimers and "mature" monomers of COL-19::GFP (Fig 2C) . 148
To examine possible involvement of tmem-39 in collagen gene transcription, we used 149
RNAi to knock-down tmem-39 in animals with the col-19p::GFP transcriptional reporter 150 in which GFP expression is driven by the promoter of col-19. In contrast to the striking 151 decrease of overall COL-19::GFP protein abundance, the transcriptional activity of the 152 col-19 promoter was not affected by tmem-39 (Figs 2B and 2D). We also evaluated 153 the mRNA level of col-19 by quantitative reverse transcription polymerase chain 154 reaction (qRT-PCR) and found that the dma258 mutant displayed a mild increase of 155 col-19 mRNA level, likely caused by compensatory feedback regulation of collagen 156 gene transcription (Fig 2E) . The dma258 mutant fully recapitulated the tmem-39 RNAi 157 phenotype in defective COL-19::GFP secretion ( Fig 2F) . 158
There are two main collagen-enriched tissues in C. elegans, the cuticle (exoskeleton) 159 and basement membranes (25). tmem-39 RNAi had no effect on the production of 160 mCherry-tagged EMB-9 (30), a Collagen IV α1 on basement membranes (S2T Fig and  161 S3 Table) . We found that loss of tmem-39 specifically affected collagens in cuticle, as 162 exemplified by LON-3::GFP and COL-101::GFP (Figs 2G-H and S3). Furthermore, 163 electron microscopy (EM) analysis revealed striking reduction of cuticle thickness in 164 dma258 mutants than in wild type ( Fig 2I) . We also noticed that TMEM-39 deficient 165 animals were small in size and dumpy, more sensitive to cuticle-disrupting osmotic 166 stresses and developed more slowly. Taken together, these results demonstrate 167 essential roles of TMEM-39 in collagen secretion, proper cuticle formation and 168 preventing ER stress likely induced by premature collagen accumulation in C. elegans. between human Sec23A C-termini with human wild type and YR mutant TMEM39A 220 cytoplasmic loop domain. 221
Predicted by the TOPCONS program, TMEM-39 contains putatively eight 223 transmembrane segments and two large cytoplasmic loops ( Fig 4B) . We further used 224 the Y2H screen to search for human proteins that could interact with the conserved 225 first loop domain (198-298 a.a.) and the second loop domain (337-420 a.a.) of 226 TMEM39A ( Fig 4C) . Among the prey cDNA clones identified from the Y2H screen, 227
Sec23A was confirmed to interact with the second loop domain of TMEM39A (Fig 4D) . The disease manifests with skeletal abnormalities, dysmorphic facial features and 235 calvarial hypomineralization, features thought to result from defects in collagen 236 secretion (37). Consistent with recent studies using the CoIP assay to demonstrate 237 association between TMEM39A and Sec23A (28), we found that TMEM39A interacted 238 with Sec23A but not Sec24D in Y2H assays (Figs 4D-E). These results indicate that 239 the TMEM39A cytoplasmic loop domain interacts specifically with Sec23A, which forms 240 an inner-shell heterodimer with Sec24 to drive procollagen secretion. 241 We next examined the loss-of-function phenotype of sec-23. RNAi knock-down of sec-242 23, the C. elegans homolog of Sec23A, strongly reduced COL-19::GFP secretion in 243 the cuticle and increased its aggregation in the intracellular region of hypoderm (Fig  244   4F ). RNAi of sec-23 also led to strong hsp-4p::GFP induction, indicating constitutively 245 activated ER stress response ( Fig 4G) 
conserved among all examined species from invertebrates to vertebrates (S1 Fig). To 261 test whether the conserved YR motif is important for interaction with Sec23A, we 262 substituted the YR motif of TMEM39A into Alanine-Alanine (AA). Using Y2H assays, 263
we found that such substitution in TMEM39A strongly attenuated its interaction with 264 Sec23A (Fig 4H) . These results show that the second cytoplasmic loop domain of 265 TMEM39A specifically binds to the COPII inner-shell component Sec23A and its C. 266 elegans homolog sec-23 is also essential for collagen production in vivo. 267
The collagen secretion phenotype of tmem-39 is independent of ER stress and 268 autophagy 269 We identified both tmem-39 and tmem-131 from the genome-wide screen for RNAi 270 clones affecting the abundance of asp-17p::GFP, which is downregulated by ER stress 271 (22). We examined collagen secretion phenotypes of other genes involved in protein 272 modification and homeostasis in the ER identified from the asp-17p::GFP screen, 273 including ostb-1, nus-1, stt-3, dlst-1, ost-3 and uggt-1 (Fig 5A and S4 Table) . RNAi RNAi, but not sac-2 RNAi, caused a marked up-regulation of the autophagy 303 transcriptional reporter tts-1p::GFP (Fig 6A) . tts-1 is a long non-coding RNA that 304 represses protein synthesis and is activated by HLH-30/TFEB, a master transcriptional 305 regulator of autophagy (43, 44). However, sac-1 RNAi did not affect the ER stress 306 response reporter hsp-4p::GFP (Fig 6B) or COL-19::GFP (Figs 6C-D) . We also 307 examined RNAi phenotypes of let-363, which encodes an ortholog of human mTOR 308 (mechanistic target of rapamycin kinase) and regulates autophagy in C. elegans (45, 309 46). Similarly as sac-1 RNAi, let-363 knock-down in C. elegans showed a marked 310 induction of tts-1p::GFP but has no apparent effects on collagen secretion (Figs 6 E-311 G). Together, these findings indicate that roles of C. elegans TMEM-39 in collagen 312 secretion are independent of ER stress response and autophagy regulation. COPII/TRAPPIII complexes for sequential ER-to-Golgi cargo transport (Fig 7) . 332 333 The second cytoplasmic loop domain of TMEM39A interacts with the core COPII 334 coating component Sec23A. TMEM131 binds to COL1A2 to facilitate assembly of 335 procollagen trimers and TRAPP III activation of Rab GTPase, in coordination with 336 TMEM39A to promote the ER-to-Golgi transport of procollagen cargo in COPII. Uso1 337 interacts with the COPII vesicle to promote targeting to the Golgi apparatus. 338
By yeast-two-hybrid assays, we found that the TMEM39A cytoplasmic loop domain can 339 interact with the Sec23A. RNAi knock-down of sec-23 and most other COPII genes 340 recapitulated the tmem-39 loss-of-function phenotypes in constitutively high ER stress 341 response, defective collagen secretion and sensitivity to osmolality stress in C. elegans 342 (Table 1 ). We also noticed that RNAi knock-down of many COPII related genes, such 343 as sec-23, sec-24.1, npp-20, sar-1, sec-12, rab-5 and trpp-8 caused more severe 344 phenotypes than tmem-39 RNAi, leading to lethality or developmental arrest that 345 prevent collagen phenotype analysis (Table 1) Recent work showed that TMEM39A facilitates the ER-to-Golgi transport of SAC1 and 355 regulates autophagosome formation (28). We found that RNAi knock-down of 356 autophagy related genes, such as sac-1 and let-363, caused autophagy induction but 357 did not affect the ER stress response or collagen secretion (Fig 6) . Genes identified 358 from the asp-17p::GFP screen that regulate the ER stress response also did not affect 359 collagen secretion (S4 Table) , further supporting the notion that roles of TMEM-39 in 360 collagen secretion are independent of ER stress response and autophagy. 361 Besides Sec23A, additional interactors were identified from Y2H screens with the 362 TMEM39A cytoplasmic domain as bait. We verified the interaction between full-length 363 DCTN6 (1-190 a.a.) and TMEM39A (337-420 a.a.) (Fig 4D) . DCTN6 is a subunit of the 364 dynactin protein complex (47) that acts as an essential cofactor of the cytoplasmic 365 dynein motor to transport a variety of cargos and organelles along the microtubule-366 based cytoskeleton (48, 49). In mammalian cells, ER-to-Golgi transport proceeds by 367 cargo assembly into COPII-coated ER export sites (ERES) followed by 368 vesicular/tubular transport along microtubule tracks toward the Golgi in a 369 dynein/dynactin-dependent manner (50). Sec23p directly interacts with the dynactin 370 complex (50), indicating that TMEM39A may participate in a Sec23/DCTN6 complex 371 to facilitate COPII coat assembly and subsequent dynein/dynactin-dependent 372 transport. Test of this hypothetic model and determination of the underlying mechanism 373 in relation to TMEM131's role in collagen secretion await further investigations. 374
Mammalian genomes encode two TMEM39 family proteins, TMEM39A and TMEM39B. 375 TMEM39A is a susceptibility locus associated with various autoimmune diseases and 376 highly up-regulated in brain tumors (33, 51). TMEM39B was recently found to interact 377 with the SARS-CoV-2 ORF9C protein, which localizes to ER-derived vesicles (52, 53). 
Hela cells were seeded in 24-well plates with cover glass, each with three replicates 468 