Endoplasmic reticulum–to–Golgi trafficking of procollagen III via conventional vesicular and tubular carriers

Abstract

Collagen is the major protein component of the extracellular matrix. Synthesis of procollagens starts in the endoplasmic reticulum (ER), and three ⍺ chains form a rigid triple helix 300–400 nm in length. It remains unclear how such a large cargo is transported from the ER to the Golgi apparatus. In this study, to elucidate the intracellular transport of fibril-forming collagens, we fused cysteine-free GFP to the N-telopeptide region of procollagen III (GFP-COL3A1) and analyzed transport by live-cell imaging. We found that the maturation dynamics of procollagen III were largely different from those of network-forming procollagen IV (Matsui et al. 2020). Proline hydroxylation of procollagen III uniquely triggered the formation of intralumenal droplet-like structures similar to events caused by liquid–liquid phase separation, and ER exit sites surrounded large droplets containing chaperones. Procollagen III was transported to the Golgi apparatus via vesicular and tubular carriers containing ERGIC53 and RAB1B; this process required TANGO1 and CUL3, which we previously reported were dispensable for procollagen IV. GFP-COL3A1 and mCherry-⍺1AT were co-transported in the same vesicle. Based on these findings, we propose that shortly after ER exit, enlarged carriers containing procollagen III fuse to ERGIC for transport to the Golgi apparatus by conventional cargo carriers. Movie S1: Live-cell imaging of COS7 cells transiently expressing GFP-COL3A1 (green) and Golgi-BFP (blue) after addition of ascorbic acid (Figure 1D). Time-lapse images were acquired every 30 min using fluorescence microscopy (Leica). The arrow indicates the Golgi apparatus. Scale bar, 10 μm. Download Original Video (2.3 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e21-07-0372/20220104/media/mc-e21-07-0372-s02.,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c7087751841cabb28b19a3e23570e4794628d00a388fabd4f47061f4b4b81c11dac4c336cd5be6a2a6a495e5def8479aeedf32e6f4ec8f325c8354b92f9a15e63bba6a844857b79b5251ea2f2c474db882c931237fd69b3bd99f9c1261a70275b889ff2377a28b4394592b4b8b9dba9521c9ff2694bc9692c8e1ede08685e57c1a49b323518a78dc4e7f9773f94f53fe7e96de7a00108e742275c59c6715abe6ec396a57845fadfdfba76b Movie S2: Live-cell imaging of RD cells transiently expressing GFP-COL3A1 (green) and PDI-mCherry (red) (Figure 2A). Time-lapse images were obtained every 5 sec using a confocal microscope (Leica SP8) ∼ 1 h after addition of ascorbic acid. The arrow indicates a GFP-COL3A1-containing vesicle trafficking from the ER to the Golgi. Scale bar, 2 μm. Download Original Video (1.0 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e21-07-0372/20220104/media/mc-e21-07-0372-s03.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c384a30613cc9136b305abcd987bb852e48c3bfa2f16ca4ee4aad0ae92a7d2e5f8643b4e08c9679052ba0208530d29f87ba2b6b53a6539009a288e1aae21665e46dca1812f27e914991221124ce60b25e82b76aea503b25 Movie S3: Living-cell imaging of RD cells transiently expressing GFP-α1AT (green) by confocal microscopy (Figure S4). Time-lapse images were obtained every 4 sec. The arrow indicates a GFP-α1AT-containing vesicle trafficking from the ER to the Golgi. Scale bar, 3 μm. Download Original Video (1.8 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e21-07-0372/20220104/media/mc-e21-07-0372-s04.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c384a30613cc9136b305abcd987bb852e48c3bfa2f16ca4ee4aad0ae92a7d2e5f8643b4e08c9679052ba0278530d29f87ba2b403120024db18364711f7f0d5f243498270c08a1490b024d43c15f3dadd5c08feffa23061c Movie S4: Time-lapse imaging of RD cells transiently expressing GFP-α1AT (green) and mScarlet-RAB1B (red) acquired by confocal microscopy at 24 h after transfection (Figure S5A). Time-lapse images were acquired every 0.5 sec. The arrow indicates a vesicle containing both GFP-α1AT and mScarlet-RAB1B. Scale bar, 2 μm. Download Original Video (5.1 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e21-07-0372/20220104/media/mc-e21-07-0372-s05.,1920,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518b17abb28b8c6166875a97f369b6d543b680497f5bca01d1632f390ebb6e08bd2a2725a70a05738b03ddc7251bffb1206bcd384e738aacf0abe9b4502e102dfa92763a3db1ea5002610ecd6cf45afbe7326f9a386c1d36f0006d1d81b9b5373201812c79db53fcc660bf6b78f30e5c80e5fb44772a3d8f0a70a1ca849de9a5cadbec9762789278d8990160e0dbd92d6961c22181787b18f34a08f23dc11b2f22ff9733aa7d46839ec4a40290 Movie S5: Live-cell imaging of RD cells transiently expressing GFP-COL3A1 (green) and mScarlet-RAB1B (red) by confocal microscopy (Figure 3A). Time-lapse images were acquired every 4 sec starting ∼1 h after addition of ascorbic acid. The arrow indicates a vesicle containing both GFP-COL3A1 and mScarlet-RAB1B. Scale bar, 2 μm. Download Original Video (2.1 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e21-07-0372/20220104/media/mc-e21-07-0372-s06.,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518417abb28b5655d276a5eae1bfc19592cc1f1a12ac8d96d0769beebf64b7a7aef1609cbd3a33d54a00c50ff29123db1c37b9b21c021889513e583236cdaa7986fef64374cd452b321552333924b207bc9dc72b7b034093b6c0b7a34856c763afc5b0f3aba405c314d59e2567811fc5b04f3b22a58bd50d6aea80cebbf595289656fc9da755ac39325233a1d95baf6f49f7789ed94903935113bc7c2c427253987c06 Movie S6: Live-cell imaging of RD cells transiently expressing GFP-COL3A1 (green) and mScarlet-RAB1A (red) by confocal...