TANGO1/cTAGE5 receptor as a polyvalent template for assembly of large COPII coats

Abstract

The supramolecular cargo procollagen is loaded into coat protein complex II (COPII)-coated carriers at endoplasmic reticulum (ER) exit sites by the receptor molecule TANGO1/cTAGE5. Electron microscopy studies have identified a tubular carrier of suitable dimensions that is molded by a distinctive helical array of the COPII inner coat protein Sec23/24•Sar1; the helical arrangement is absent from canonical COPII-coated small vesicles. In this study, we combined X-ray crystallographic and biochemical analysis to characterize the association of TANGO1/cTAGE5 with COPII proteins. The affinity for Sec23 is concentrated in the proline-rich domains (PRDs) of TANGO1 and cTAGE5, but Sec23 recognizes merely a PPP motif. The PRDs contain repeated PPP motifs separated by proline-rich linkers, so a single TANGO1/cTAGE5 receptor can bind multiple copies of coat protein in a close-packed array. We propose that TANGO1/cTAGE5 promotes the accretion of inner coat proteins to the helical lattice. Furthermore, we show that PPP motifs in the outer coat protein Sec31 also bind to Sec23, suggesting that stepwise COPII coat assembly will ultimately displace TANGO1/cTAGE5 and compartmentalize its operation to the base of the growing COPII tubule.vesicle traffic | coat protein | procollagen T he coat protein complex II (COPII)-coated vesicles transport secretory and plasma membrane proteins from the endoplasmic reticulum (ER). COPII vesicle budding involves a stepwise assembly reaction: Membrane-bound Sar1-GTP recruits Sec23/24 to form the inner coat complex that, in turn, recruits the Sec13/31 outer coat protein (1). Sec13/31 self-assembles into a polyhedron, and in the process, it sculpts the ER membrane into a bud, producing a COPII-coated vesicle with a diameter of ∼60 nm (2-4).Small cargo molecules are captured during the budding reaction through mechanisms that are well established (5). However, the formation of canonical 60-nm COPII vesicles cannot explain the packaging of large cargos, such as the procollagen fibril with a length of 300-400 nm. Procollagen follows the conventional route of secretion taken by other extracellular proteins, and its ER export evidently depends on COPII carriers because mutations in genes encoding COPII proteins result in collagen secretion blockade, impaired extracellular matrix deposition, and abnormal craniofacial development (6-8). A key discovery was the identification of the receptor TANGO1 and its coreceptor cTAGE5 as ER-localized machinery for loading procollagen into COPII carriers (9, 10). TANGO1 has a luminal SH3 domain shown to interact with collagen VII and a cytosolic domain that interacts with Sec23/24. Both TANGO1 and cTAGE5 are required for collagen export from the ER (9, 10). The TANGO1 knockout mouse has generalized defects in extracellular matrix formation, owing to a block in ER export of multiple collagen types (11).Recent research has implicated post-ER membranes in TANGO1-mediated carrier formation (12, 13); however, the mechanism of action of TANGO1/cTAGE5 in procollagen export...

TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER–Golgi intermediate compartments

“…These data define the minimal domain of TFG that is necessary for its association with Sec23 ( Fig. S2G), although its proline-rich domain (PRD) may also contribute to binding, based on previous work examining PRDs from Sec31 and Tango1/cTAGE5 (40).…”

“…5C). Current evidence suggests that Tango1 acts early to organize exit sites on the ER and enable procollagen export (40,70,71), whereas TFG functions to initiate dissociation of the outer COPII coat and restrict diffusion of inner COPII-coated carriers at the ER/ERGIC interface.…”

“…Some of these interactions have been shown to be competitive, suggesting that unique associations with Sec23 occur sequentially as transport carriers form, undergo scission from the ER, and tether to ERGIC membranes (14,40). We used confocal and stimulated emission depletion (STED) microscopy to define the relative distribution of TFG with several of these factors, based on the availability of validated antibodies.…”

“…Both Sec31 and TANGO1/cTAGE5 exploit PRDs to associate with a common motif on Sec23 (40). Given the presence of a PRD in TFG, we investigated the possibility that it interacts with Sec23 in a manner that would compete with Sec31 and/or TANGO1.…”

“…Members of the Sec16 family function at ER subdomains to inhibit Sar1 GTPase activity and facilitate COPII coat assembly (36)(37)(38). In a related manner, the cargo receptors Tango1 and cTAGE5 associate directly with Sec23 at the ER to promote the formation of elongated COPII-coated tubules (39,40). Ultimately, outer coat assembly has been suggested to outcompete Tango1/cTAGE5 and Sec16 interactions with the inner coat to enable fission of transport carriers in a manner dependent on Sar1 GTP hydrolysis (40,41).…”