KLHL12 can form large COPII structures in the absence of CUL3 neddylation.
KLHL12 can form large COPII structures in the absence of CUL3 neddylation.
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DOI：
10.1091/mbc.e22-08-0383
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发表时间：
2023-03-01
影响因子：
3.3
通讯作者：
Kim, Jinoh
中科院分区：
文献类型：
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作者：
Moretti, Tamara;Kim, Kyungho;Tuladhar, Astha;Kim, Jinoh
CUL 3-RING泛素连接酶（CRL 3）通过不同的Bric-a-brac、Tramtrack和Broad-complex（BTB）结构域蛋白参与各种细胞过程。KLHL 12是一种BTB结构域蛋白，被认为在从内质网（ER）输出大货物分子（如前胶原）中发挥重要作用。CRL 3 KLHL 12使SEC 31单倍化，导致COPII囊泡尺寸增加。扩大的COPII囊泡可以容纳前胶原分子。因此，CRL 3 KLHL 12对于大COPII结构的组装和胶原蛋白分泌是必需的。CRL 3通过CUL 3 neddylation激活。在此，我们评估了CUL 3 neddylation在COPII组装和胶原分泌中的重要性。出乎意料的是，用MLN 4924（一种NEDD 8激活酶的强效抑制剂）处理后，大COPII-KLHL 12结构的组装持续存在，细胞胶原蛋白水平降低。当我们在CUL 3界面处将突变引入KLHL 12时，这些KLHL 12变体不与neddylated CUL 3相互作用，但其中一个（Mut A）仍然支持大的COPII-KLHL 12结构。野生型KLHL 12的过表达，而不是Mut A，降低细胞胶原蛋白水平最有可能通过溶酶体降解。我们的结果表明，CUL 3 neddylation对于形成大的COPII-KLHL 12结构来说不是必需的，但活性CRL 3 KLHL 12有助于维持细胞中的胶原蛋白水平。
CUL3-RING ubiquitin ligases (CRL3s) are involved in various cellular processes through different Bric-a-brac, Tramtrack, and Broad-complex (BTB)–domain proteins. KLHL12, a BTB-domain protein, is suggested to play an essential role in the export of large cargo molecules such as procollagen from the endoplasmic reticulum (ER). CRL3KLHL12 monoubiquitylates SEC31, leading to an increase in COPII vesicle dimension. Enlarged COPII vesicles can accommodate procollagen molecules. Thus, CRL3KLHL12 is essential for the assembly of large COPII structures and collagen secretion. CRL3s are activated by CUL3 neddylation. Here, we evaluated the importance of CUL3 neddylation in COPII assembly and collagen secretion. Unexpectedly, the assembly of large COPII-KLHL12 structures persisted and cellular collagen levels decreased on treatment with MLN4924, a potent inhibitor of NEDD8-activating enzyme. When we introduced mutations into KLHL12 at the CUL3 interface, these KLHL12 variants did not interact with neddylated CUL3, but one of them (Mut A) still supported large COPII–KLHL12 structures. Overexpression of wild-type KLHL12, but not Mut A, lowered cellular collagen levels most likely via lysosomal degradation. Our results suggest that CUL3 neddylation is not necessary for the formation of large COPII–KLHL12 structures, but active CRL3KLHL12 contributes to the maintenance of collagen levels in the cell.