Abstract
Large coat protein complex II (COPII)-coated vesicles serve to convey the large cargo procollagen I (PC1) from the endoplasmic reticulum (ER). The link between large cargo in the lumen of the ER and modulation of the COPII machinery remains unresolved. TANGO1 is required for PC secretion and interacts with PC and COPII on opposite sides of the ER membrane, but evidence suggests that TANGO1 is retained in the ER, and not included in normal size (<100 nm) COPII vesicles. Here we show that TANGO1 is exported out of the ER in large COPII-coated PC1 carriers, and retrieved back to the ER by the retrograde coat, COPI, mediated by the C-terminal RDEL retrieval sequence of HSP47. TANGO1 is known to target the COPII initiation factor SEC12 to ER exit sites through an interacting protein, cTAGE5. SEC12 is important for the growth of COPII vesicles, but it is not sorted into small budded vesicles. We found both cTAGE5 and SEC12 were exported with TANGO1 in large COPII carriers. In contrast to its exclusion from small transport vesicles, SEC12 was particularly enriched around ER membranes and large COPII carriers that contained PC1. We constructed a split GFP system to recapitulate the targeting of SEC12 to PC1 via the luminal domain of TANGO1. The minimal targeting system enriched SEC12 around PC1 and generated large PC1 carriers. We conclude that TANGO1, cTAGE5, and SEC12 are copacked with PC1 into COPII carriers to increase the size of COPII, thus ensuring the capture of large cargo.
Highlights
Large coat protein complex II (COPII)-coated vesicles serve to convey the large cargo procollagen I (PC1) from the endoplasmic reticulum (ER)
In search of a mechanism to explain the genetic requirement for COPII to secrete large cargo-like PC1 [9, 10], we had previously discovered the existence of large COPII vesicles and established them as bona fide carriers of PC1 [11, 12]
We examined their molecular composition and discovered that TANGO1, cTAGE5, and SEC12 are copackaged with PC1 into COPII carriers (Figs. 1 and 4)
Summary
TANGO1, cTAGE5, and SEC12 Are Copackaged into Large COPII Carriers Along with PC1. The large transmembrane protein TANGO1 is poised to be a COPII receptor for large PC cargo as it interacts with COPII and PC on opposite sides of the ER membrane (Fig. 1A) [14, 16, 18,19,20]. TANGO1 accumulated around PC1 puncta and colocalized with the COPII outer coat protein SEC31A (Fig. 3 C and D) These exceptionally large COPII-decorated membranes were much bigger than the functional carriers we observed by STORM and CLEM [11], and were readily resolved by confocal microscopy. The SEC12/PC1 flat discs possibly represented PC1-containing ERES before the recruitment and activation of SAR1 (Fig. 5 C, i), which would explain why little SEC31A was observed overlapping with SEC12, given that the SEC13/31 outer coat is recruited after the activation of SAR1 and the recruitment of the inner coat [28] These 3D-STORM data supported our biochemical analyses of large COPII-coated PC1 carriers generated in a cell-free reaction. GFP11 (the 11th β-strand of GFP) was fused to the C terminus of 3xFLAG-SEC12 so that it would be exposed on the short ER luminal tail of SEC12; GFP1-10 (the rest of GFP without the 11th β-strand) was fused to the C terminus of the luminal SH3 and PNAS | vol 115 | no. 52 | E12259
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