A COMMON UBIQUITIN-PROTEASOME DEGRADATION PATHWAY FOR TAIL-ANCHORED RECEPTORS OF TOC AND TOM COMPLEXES IN ARABIDOPSIS THALIANA

Chloroplasts and mitochondria are two important organelles in plant cells. Thousands of nucleus-encoded proteins are imported into chloroplasts and mitochondria via the translocons at the outer membrane of chloroplasts (TOC) and mitochondria (TOM), respectively. Proteolytic regulation of TOC and TOM complexes is critically important for organellar function and plant physiology, but the understanding of these processes is minimal. In this study, the biological function of an uncharacterized Arabidopsis thaliana (Arabidopsis) protein encoded by At5g42220 was investigated. This cytosolic protein specifically interacted with the transmembrane domain (TMD) of TOC and TOM receptors in the cytosol of plant cells and was, therefore, named TMD-binding protein for Tail-anchored Outer membrane Proteins, TTOP. Knock-out of TTOP did not affect plant growth but impaired chloroplast biogenesis in de-etiolation experiments, indicating a role of TTOP in chloroplast biogenesis. However, overexpression of TTOP induced plant chlorosis and was lethal. Such phenotypes were caused by the removal of the TOC complex, which could lead to chloroplast degeneration or the impairment of chloroplast biogenesis. Bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation (co-IP) analysis confirmed the interactions between TTOP and components of chloroplast-associated protein degradation (CHLORAD) system, including SP1 (suppressor of ppi1 locus1), CDC48A (cell division cycle 48), and 26S proteasome (26SP) subunits, suggesting that TTOP was involved in the CHLORAD system. The CHLORAD is a proteolytic system that degrades the TOC apparatus. In this process, the E3 ligase (Ubiquitin ligating enzyme) SP1 on the OEM ubiquitinates TOC proteins, which is followed by the action of SP2 (suppressor of ppi1 locus2) and CDC48A as a conduit and a motor, respectively, for retrotranslocation of TOC complex out of the OEM. Upon its release into the cytosol, the ubiquitinated substrates are delivered to the 26SP for degradation, possibly via unknown cytosolic factors. The blue native polyacrylamide gel electrophoresis (BN-PAGE) results confirmed the co-existence of TTOP, Toc33, and CDC48A, or TTOP, Toc33, and 26SP subunit in one complex in vivo, suggesting that TTOP is a cytosolic cofactor for the delivery of the ubiquitinated TOC proteins to the 26SP. In yeast, Cdc48 mediates the retrotranslocation of mitochondrial OEM proteins for their proteasomal degradation, and it is also recruited to the TOM translocons for retrotranslocation and removal of precursor proteins that are clogged in the TOM translocons. In this study, CDC48A also interacted with the TOM complex on mitochondrial OEM, and a co-localization of CDC48A, TOM proteins, and TTOP was shown to surround mitochondria in plant cells. In addition, BN-PAGE analysis showed the co-existence of TTOP, Tom20-2, and CDC48A, or TTOP, Tom20-2, and 26SP subunit in one complex in vivo, suggesting that the mechanism for TOM complex clearance might be similar to the CHLORAD pathway for TOC complex turnover in plants. Together, this suggests the existence of a common proteasome-dependent proteolysis mechanism for both TOC and TOM receptors, in which CDC48A and TTOP act as a motor for the retrotranslocation of substrates and a cytosolic shuttling cofactor for proteasomal substrate delivery, respectively.

叶绿体和线粒体是植物细胞中的两个重要细胞器。绝大部分的叶绿体和线粒体蛋白由细胞核编码，再通过叶绿体和线粒体外膜的运输蛋白复合体，分别是TOC 和TOM运输进入叶绿体和线粒体。对TOC和TOM复合物的蛋白降解的调节对于细胞器功能和植物生理学至关重要，但对这些过程的了解很少。 在这项研究中，主要研究了由拟南芥 (Arabidopsis) At5g42220基因编码的蛋白质的生物学功能。该胞质蛋白与植物细胞胞质中的TOC和TOM受体的跨膜结构域 (TMD) 有特异性相互作用，因此被命名为尾锚定外膜蛋白的 TMD 结合蛋白，TTOP。TTOP的敲除不影响植物生长，但在去黄体化实验中抑制了叶绿体合成，表明TTOP参与了叶绿体合成。然而，TTOP的过度表达会诱导植物萎黄并且致死。这种黄化并致死的表型是由去除了叶绿体上的TOC复合物引起的，这可能导致叶绿体降解或抑制叶绿体合成。 双分子荧光互补 (BiFC) 和共免疫沉淀 (co-IP) 分析证实了TTOP与叶绿体相关蛋白降解途径 (CHLORAD)的成分之间有相互作用，其成本包括SP1、CDC48A和26S蛋白酶体 (26SP)亚基，表明 TTOP 参与了 CHLORAD 系统。 CLORAD 是一条降解TOC蛋白复合体的蛋白水解系统。在该降解过程中，叶绿体外膜上的 E3 连接酶（泛素连接酶）SP1 泛素化 TOC蛋白，随后 SP2和 CDC48A 分别作为管道和马达将 TOC 复合物的逆向易位出外膜。在TOC蛋白其释放到细胞质中后，再通过未知的细胞质因子被递送至 26SP 进行降解。蓝色天然聚丙烯酰胺凝胶电泳 (BN-PAGE) 结果证实 TTOP、Toc33 和 CDC48A 或 TTOP、Toc33 和 26SP 亚基共存于同一个复合物中，表明 TTOP 是作为一种胞质辅因子泛素化的TOC 蛋白传递到26SP 进行降解。 在酵母中，Cdc48 介导线粒体外膜蛋白的逆向易位以使其通过蛋白酶体降解，并且它还被招募到 TOM受体从而逆向易位和去除堵塞在 TOM 受体中的前体蛋白。在这项研究中，CDC48A 同时与线粒体外膜上的 TOM 复合物相互作用，并且结果表明CDC48A、TOM 蛋白和 TTOP 的有共定位且其共定位在线粒体外围。此外，BN-PAGE 分析表明 TTOP、Tom20-2 和 CDC48A 或 TTOP、Tom20-2 和 26SP 亚基共存于体内同一个复合物中，表明 TOM 复合物降解的机制可能类似于将TOC复合物降解的 CHLORAD 途径。这些结果表明在植物细胞中存在共同的蛋白酶体参与的TOC 和 TOM 受体蛋白降解机制，其中 CDC48A 和 TTOP 分别作为将底物逆向易位的马达和将底物递送到蛋白酶体降解的胞质运输因子。