Laboratory of Molecular and Cellular Biology, Institute for Frontier Life and Medical Sciences, Kyoto University.
Department of Cell Sciences, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine.
Cell Struct Funct. 2020 Jul 23;45(2):107-119. doi: 10.1247/csf.20025. Epub 2020 Jun 18.
Collagen is the most abundant protein in animal tissues and is critical for their proper organization. Nascent procollagens in the endoplasmic reticulum (ER) are considered too large to be loaded into coat protein complex II (COPII) vesicles, which have a diameter of 60-80 nm, for exit from the ER and transport to the Golgi complex. To study the transport mechanism of procollagen IV, which generates basement membranes, we introduced a cysteine-free GFP tag at the N-terminus of the triple helical region of the α1(IV) chain (cfSGFP2-col4a1), and examined the dynamics of this protein in HT-1080 cells, which produce endogenous collagen IV. cfSGFP2-col4a1 was transported from the ER to the Golgi by vesicles, which were a similar size as small cargo carriers. However, mCherry-ERGIC53 was recruited to α-antitrypsin-containing vesicles, but not to cfSGFP2-col4a1-containing vesicles. Knockdown analysis revealed that Sar1 and SLY1/SCFD1 were required for transport of cfSGFP2-col4a1. TANGO1, CUL3, and KLHL12 were not necessary for the ER-to-Golgi trafficking of procollagen IV. Our data suggest that procollagen IV is exported from the ER via an enlarged COPII coat carrier and is transported to the Golgi by unique transport vesicles without recruitment of ER-Golgi intermediate compartment membranes.Key words: collagen, procollagen IV, endoplasmic reticulum, ER-to-Golgi transport, ERGIC.
胶原蛋白是动物组织中最丰富的蛋白质，对其正常组织至关重要。内质网（ER）中的新生前胶原被认为太大，无法装入直径为 60-80nm 的衣壳蛋白复合物 II（COPII）小泡，从而无法从 ER 中逸出并运输到高尔基体复合物。为了研究基底膜产生的四型前胶原的运输机制，我们在α1(IV)链的三螺旋区的 N 端引入了一个不含半胱氨酸的 GFP 标签（cfSGFP2-col4a1），并在产生内源性胶原 IV 的 HT-1080 细胞中检查了这种蛋白质的动力学。cfSGFP2-col4a1 通过与小货物载体大小相似的小泡从 ER 运输到高尔基体。然而，mCherry-ERGIC53 被募集到包含α-抗胰蛋白酶的小泡中，但不被募集到包含 cfSGFP2-col4a1 的小泡中。敲低分析表明 Sar1 和 SLY1/SCFD1 是 cfSGFP2-col4a1 运输所必需的。TANGO1、CUL3 和 KLHL12 对于前胶原 IV 的 ER 到高尔基体的运输不是必需的。我们的数据表明，前胶原 IV 通过扩大的 COPII 衣壳载体从 ER 中输出，并通过独特的运输小泡运输到高尔基体，而不招募 ER-Golgi 中间隔室膜。关键词：胶原蛋白、四型前胶原、内质网、ER 到高尔基体运输、ERGIC。
