Intracellular trafficking and degradation of unassociated proα2 chains of collagen type I
Intracellular trafficking and degradation of unassociated proα2 chains of collagen type I
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DOI：
10.1016/j.yexcr.2004.01.029
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发表时间：
2004-06-10
影响因子：
3.7
通讯作者：
Bienkowski, RS
中科院分区：
文献类型：
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作者：
Gotkin, MG;Ripley, CR;Bienkowski, RS
前胶原I是由两条前α 1（I）链和一条前α 2（I）链组成的三聚体。在某些情况下，轻度成骨不稳，异常的proalpha1（1）链在合成后很快降解。因此，细胞产生过量的前α（1）链，其不能形成三聚体并且不被分泌。这项工作的目的是确定细胞内的命运的未关联的proalpha2（I）链。Mov13小鼠成纤维细胞不合成proalpha1（1）mRNA，但产生proalpha2（1）mRNA，使用脉冲追踪方案与放射性氨基酸孵育，并通过凝胶电泳、放射自显影和Western印迹分析蛋白质。Mov13细胞产生的proalpha2（l）链，在30分钟内降解细胞内。当细胞用布雷菲德菌素A，它阻止从内质网到高尔基体的运输处理时，降解被抑制。固定的细胞暴露于各种免疫荧光标记物，并通过共聚焦激光扫描显微镜成像显示，proalpha2（l）链与高尔基体和溶酶体标记物共定位。当细胞用渥曼青霉素处理时，降解被抑制，链被分泌，渥曼青霉素阻断了向溶酶体的运输。这些结果表明，未结合的proalpha2（1）链离开内质网，通过高尔基体，进入溶酶体，在那里被降解。(C)2004年爱思唯尔公司All rights reserved.
Procollagen I is a trimer consisting of two proalpha1 (I) chains and one proalpha2(I) chain. In certain cases of mild osteogenesis imperfecta, abnormal proalpha1 (1) chains are degraded very soon after synthesis. As a consequence, the cells produce excess proalpha(1) chains, which cannot form trimers and are not secreted. The objective of this work was to determine the intracellular fate of unassociated proalpha2(I) chains. Mov13 mouse fibroblasts, which do not synthesize proalpha1 (1) mRNA, but do produce proalpha2(l) mRNA, were incubated with radioactive amino acids using pulse-chase protocols, and proteins were analyzed by gel electrophoresis, autoradiography, and Western blotting. Mov13 cells produced proalpha2(l) chains that were degraded intracellularly within 30 min. Degradation was inhibited when cells were treated with brefeldin-A, which blocks transit from endoplasmic reticulum to Golgi. Fixed cells exposed to various immunofluorescence markers and imaged by confocal laser scanning microscopy showed that proalpha2(l) chains colocalized with Golgi and lysosome markers. Degradation was inhibited and chains were secreted when cells were treated with wortmannin, which blocks trafficking to lysosomes. These results demonstrate that unassociated proalpha2(1) chains leave the endoplasmic reticulum, transit the Golgi, and enter lysosomes where they are degraded. (C) 2004 Elsevier Inc. All rights reserved.