叶绿体相关蛋白降解中UBX结构域蛋白将Cdc48募集到叶绿体中-动脉网

Recruitment of Cdc48 to chloroplasts by a UBX-domain protein in chloroplast-associated protein degradation

Nature 等信源发布 2024-08-19 23:13

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AbstractThe translocon at the outer chloroplast membrane (TOC) is the gateway for chloroplast protein import and so is vital for photosynthetic establishment and plant growth. Chloroplast-associated protein degradation (CHLORAD) is a ubiquitin-dependent proteolytic system that regulates TOC. In CHLORAD, cytosolic Cdc48 provides motive force for the retrotranslocation of ubiquitinated TOC proteins to the cytosol but how Cdc48 is recruited is unknown.

摘要叶绿体外膜（TOC）的易位子是叶绿体蛋白质输入的门户，因此对光合建立和植物生长至关重要。叶绿体相关蛋白降解（CHLORAD）是一种泛素依赖性蛋白水解系统，可调节TOC。在CHLORAD中，胞质Cdc48为泛素化TOC蛋白向胞质溶胶的逆转录提供了动力，但Cdc48是如何募集的尚不清楚。

Here, we identify plant UBX-domain protein PUX10 as a component of the CHLORAD machinery. We show that PUX10 is an integral chloroplast outer membrane protein that projects UBX and ubiquitin-associated domains into the cytosol. It interacts with Cdc48 via its UBX domain, bringing it to the chloroplast surface, and with ubiquitinated TOC proteins via its ubiquitin-associated domain.

在这里，我们将植物UBX结构域蛋白PUX10鉴定为CHLORAD机制的组成部分。我们显示PUX10是一种完整的叶绿体外膜蛋白，可将UBX和泛素相关结构域投射到细胞质中。它通过其UBX结构域与Cdc48相互作用，将其带到叶绿体表面，并通过其泛素相关结构域与泛素化的TOC蛋白相互作用。

Genetic analyses in Arabidopsis revealed a requirement for PUX10 during CHLORAD-mediated regulation of TOC function and plant development. Thus, PUX10 coordinates ubiquitination and retrotranslocation activities of CHLORAD to enable efficient TOC turnover..

拟南芥的遗传分析表明，在氯代介导的TOC功能和植物发育调节过程中，需要PUX10。因此，PUX10协调CHLORAD的泛素化和反转录活性，以实现有效的TOC周转。。

MainMost chloroplast proteins (>90%) are synthesized in the cytosol and imported into chloroplasts post-translationally. The chloroplast protein import machinery consists of two translocons, a translocon located in the outer chloroplast membrane (TOC) and a translocon in the inner chloroplast membrane (TIC).

大多数叶绿体蛋白（>90%）在细胞质中合成，并在翻译后导入叶绿体。叶绿体蛋白质导入机制由两个易位子组成，一个位于叶绿体外膜（TOC）中的易位子和一个位于叶绿体内膜（TIC）中的易位子。

Core components of the TOC are the β-barrel protein, TOC75, and the GTPases TOC159 and TOC33—all named in accordance with their molecular masses in kilodaltons. TOC75 forms a membrane channel for protein conductance, whereas TOC159 and TOC33 function as receptors by binding the transit peptides of precursor proteins via their cytosolic GTPase domains1,2,3,4,5,6,7,8.Chloroplast protein import is dynamically regulated by chloroplast-associated protein degradation (CHLORAD), a ubiquitin-dependent proteolytic system that targets the TOC apparatus9,10.

TOC的核心成分是β桶蛋白TOC75以及GTPases TOC159和TOC33，它们都是根据千道尔顿的分子量命名的。TOC75形成蛋白质电导的膜通道，而TOC159和TOC33通过其胞质GTPase结构域1,2,3,4,5,6,7,8结合前体蛋白的转运肽而起受体的作用。叶绿体蛋白进口受叶绿体相关蛋白降解（CHLORAD）的动态调节，CHLORAD是一种靶向TOC装置的泛素依赖性蛋白水解系统9,10。

By reconfiguring the TOC machinery, CHLORAD action facilitates changes in the organelle’s proteome, functions and morphology. Such CHLORAD-mediated TOC regulation enables the biogenesis and operation of chloroplasts (and of other members of the plastid family of organelles) to be responsive to developmental and environmental cues, including stress11,12,13.The first characterized CHLORAD component was the ubiquitin E3 ligase suppressor of ppi locus 1 (SP1).

通过重新配置TOC机制，CHLORAD作用促进了细胞器蛋白质组，功能和形态的变化。。

The SP1 protein is located in the chloroplast outer envelope membrane (OEM) and has a cytosol-facing RING domain and two transmembrane (TM) spans flanking an intermembrane space (IMS) domain that binds to TOC protein targets9. Analysis of sp1-mutant and SP1-overexpressor Arabidopsis plants showed that SP1 expression levels correlate inversely with the abundance of TOC proteins, resulting in the suppression or enhancement of the pale-green ppi1 (TOC33 knockout)14 mutant p.

SP1蛋白位于叶绿体外膜（OEM）中，具有面向胞质溶胶的RING结构域，两个跨膜（TM）跨越侧翼的膜间空间（IMS）结构域，该结构域与TOC蛋白靶标结合9。对sp1突变体和sp1过表达拟南芥植物的分析表明，sp1表达水平与TOC蛋白的丰度呈负相关，导致浅绿色ppi1（TOC33敲除）14突变体p的抑制或增强。

Data availability

数据可用性

All data generated or analysed during this study are included in this published article or its Supplementary Information. Gene sequences for the following proteins from A. thaliana were used experimentally in this study: PUX1 (At3g27310), PUX2 (At2g01650), PUX3 (At4g22150), PUX4 (At4g04210), PUX5 (At4g15410), PUX6 (At3g21660), PUX7 (At1g14570), PUX8 (At4g11740), PUX9 (At4g00752), PUX10 (At4g10790), PUX11 (At2g43210), PUX12 (At3g23605), PUX13 (At4g23040), SP1 (At1g63900), SP2 (At3g44160), CDC48A (At3g09840), TOC159 (At4g02510), TOC33 (At1g02280), TOC120 (At3g16620), TOC132 (At2g16640), TOC34 (At5g05000), TOC75 (At3g46740), TIC110 (At1g06950), TIC40 (At5g16620), CDKA1 (At3g48750), SFR2 (At3g06510) and ubiquitin (At4g05320).

本研究期间生成或分析的所有数据均包含在本文或其补充信息中。本研究通过实验使用了拟南芥中以下蛋白质的基因序列：PUX1（At3g27310），PUX2（At2g01650），PUX3（At4g22150），PUX4（At4g04210），PUX5（At4g15410），PUX6（At3g21660），PUX7（At1g14570），PUX8（At4g11740），PUX9（At4g00752），PUX10（At4g10790），PUX11（At2g43210），PUX12（At3g23605），PUX13（At4g23040），SP1（At1g63900），SP2（AT4G43210）At3g44160），CDC48A（At3g09840），TOC159（At4g02510），TOC33（At1g02280），TOC120（At3g16620），TOC132（At2g16640），TOC34（At5g05000），TOC75（At3g46740），TIC110（At1g06950），TIC40（At5g16620），CDKA1（At3g48750），SFR2（At3g06510）和泛素（At4g05320）。

Amino acid sequences of the UBX domains of the following proteins from different species were used in this study: Oryza sativa Os10g37630 (AAP54662), Zea mays GRMZM2G159538 (AQL10361), Marchantia polymorpha Mapoly0001s0291 (PTQ50274), Chlamydomonas reinhardtii Cre03.g200100 (A0A2K3DZI1), Saccharomyces cerevisiae Ubx2 (Q04228) and Homo sapiens UBXD8/FAF2 (Q96CS3).

本研究使用了来自不同物种的以下蛋白质的UBX结构域的氨基酸序列：水稻Os10g37630（AAP54662），玉米GRMZM2G159538（AQL10361），Marchantia polymorpha Mapoly0001s0291（PTQ50274），莱茵衣藻Cre03.g200100（A0A2K3DZI1），酿酒酵母Ubx2（Q04228）和智人UBXD8/FAF2（Q96CS3）。

Sequences were obtained from the TAIR (https:// www.arabidopsis.org/), Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html), Ensembl Plants (https://plants.ensembl.org/index.html), Uniprot (https://www.uniprot.org/) or National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) databases.

序列来自TAIR（https://www.arabidopsis.org/），Phytozome(https://phytozome.jgi.doe.gov/pz/portal.html)，Ensembl植物(https://plants.ensembl.org/index.html)，Uniprot(https://www.uniprot.org/)或国家生物技术信息中心(https://www.ncbi.nlm.nih.gov/)数据库。

Source data are provided with this paper..

本文提供了源数据。。

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Download referencesAcknowledgementsWe thank Q. Ling for helping to initiate the study and for many discussions during the course of the work. We thank E. Johnson and C. Melia for TEM conducted in the Sir William Dunn School of Pathology EM Facility, Z. Lewis for initiating the subcellular localization screening of PUX proteins, N.

下载参考文献致谢我们感谢Q.Ling帮助启动了这项研究，并在工作过程中进行了许多讨论。我们感谢E.Johnson和C.Melia在William Dunn病理学院EM设施Z.Lewis进行的TEM启动了PUX蛋白的亚细胞定位筛选。

Buayam for technical support with LD staining and confocal imaging, N. G. Irani and I. Moore for the organelle marker lines, F. Homma for assistance with the structural analysis, and P. Bota and J. Bateman for technical assistance. This work was supported by grants from UK Research and Innovation–Biotechnology and Biological Sciences Research Council (UKRI-BBSRC; grant numbers BB/K018442/1, BB/N006372/1, BB/R016984/1, BB/R009333/1, BB/V007300/1, BB/W015021/1 and BB/X000192/1) to R.P.J.

Buayam为LD染色和共聚焦成像提供技术支持，N.G.Irani和I.Moore为细胞器标记系，F.Homma为结构分析提供帮助，P.Bota和J.Bateman为技术援助。这项工作得到了英国研究与创新-生物技术和生物科学研究委员会（UKRI-BBSRC；资助号BB/K018442/1，BB/N006372/1，BB/R016984/1，BB/R009333/1，BB/V007300/1，BB/W015021/1和BB/X000192/1）对R.P.J的资助。

and by a PhD studentship from the Oxford Interdisciplinary Bioscience Doctoral Training Partnership (UKRI-BBSRC grant number BB/M011224/1) to N.L. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission.Author informationAuthors and AffiliationsSection of Molecular Plant Biology, Department of Biology, University of Oxford, Oxford, UKNa Li & R.

并且由牛津跨学科生物科学博士培训合作伙伴关系（UKRI-BBSRC资助号BB/M011224/1）向N.L.的博士研究生。为了开放获取的目的，作者已经向任何作者接受的稿件（AAM）版本申请了CC公共版权许可证。作者信息作者和附属机构牛津大学生物系分子植物生物学系，UKNa Li＆R。

Paul JarvisAuthorsNa LiView author publicationsYou can also search for this author in.

PaulJarvisAuthorsnaLiview作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsN.L. designed and conducted the experiments, analysed the data and wrote the article. R.P.J. conceived of the study, supervised the work, analysed the data and wrote the article.Corresponding authorCorrespondence to

PubMed谷歌学术贡献。五十、设计并进行了实验，分析了数据并撰写了文章。R、 P.J.构思了这项研究，监督了这项工作，分析了数据并撰写了这篇文章。对应作者对应

R. Paul Jarvis.Ethics declarations

R、保罗·贾维斯。道德宣言

Competing interests

相互竞争的利益

The application of CHLORAD as a technology for crop improvement is covered by a patent application (number WO2019/171091 A). The authors declare no other competing interests.

专利申请（编号WO2019/171091A）涵盖了氯拉德作为作物改良技术的应用。作者声明没有其他利益冲突。

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Nature Plants thanks the anonymous reviewers for their contribution to the peer review of this work.

Nature Plants感谢匿名审稿人对这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Subcellular localization analysis of Arabidopsis PUX proteins by confocal microscopy.Protoplasts transiently expressing YFP-tagged PUX proteins under the constitutive 35S promoter were analysed by confocal microscopy.

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。扩展数据扩展数据图1通过共聚焦显微镜对拟南芥PUX蛋白进行亚细胞定位分析。通过共聚焦显微镜分析在组成型35S启动子下瞬时表达YFP标记的PUX蛋白的原生质体。

Representative protoplasts are presented. Exposure times and gain settings were identical. Scale bar = 20 µm. Note that PUX14, PUX15 and PUX16 were designated as pseudogenes in a previous report, based on the presence of frameshift and nonsense mutations, and so were excluded from this analysis26.Extended Data Fig.

介绍了代表性的原生质体。曝光时间和增益设置是相同的。比例尺=20微米。请注意，基于移码突变和无义突变的存在，PUX14，PUX15和PUX16在先前的报告中被指定为假基因，因此被排除在本分析之外26。

2 Localization of PUX10 in chloroplasts in transgenic plants.Constructs encoding PUX10-YFP driven by the native PUX10 promoter (pPUX10) or by the constitutive 35S promoter (p35S) were used for stable plant transformation. Rosette leaves taken from 28-day-old T1 transgenic plants were visualized by confocal microscopy.

2 PUX10在转基因植物叶绿体中的定位。由天然PUX10启动子（pPUX10）或组成型35S启动子（p35S）驱动的编码PUX10-YFP的构建体用于稳定的植物转化。通过共聚焦显微镜观察来自28天大的T1转基因植物的莲座叶。

Representative images are presented. Similar localization of the YFP signals was observed in 5 to 10 independent T1 transgenic plants. Exposure times and gain settings were identical. Scale bars = 20 µm.Extended Data Fig. 3 Molecular and phenotypic characterization of two pux10 T-DNA insertion mutants.a, Schematic representation of the Arabidopsis PUX10 genomic locus (At4g10790), annotated with the positions of the pux10 T-DNA insertion mutations.

呈现了代表性的图像。在5至10个独立的T1转基因植物中观察到类似的YFP信号定位。曝光时间和增益设置是相同的。比例尺=20μm。扩展数据图3两个pux10 T-DNA插入突变体的分子和表型表征。a，拟南芥pux10基因组基因座（At4g10790）的示意图，用pux10 T-DNA插入突变的位置注释。

The positions of PCR primers used in b are also indicated, with arrows. Black boxes show exons, interconnecting white boxes show introns, and grey boxes show untranslated regions. Abbreviations: LB, left border sequences of the SAIL and Wisconsin T-DNA insertions; ATG, translation initiation codon; St.

。黑框显示外显子，相互连接的白框显示内含子，灰框显示非翻译区。缩写：LB，SAIL和威斯康星州T-DNA插入的左边界序列；ATG，翻译起始密码子；圣。

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

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Reprints and permissionsAbout this articleCite this articleLi, N., Jarvis, R.P. Recruitment of Cdc48 to chloroplasts by a UBX-domain protein in chloroplast-associated protein degradation.

转载和许可本文引用本文Li，N.，Jarvis，R.P。在叶绿体相关蛋白降解中，UBX结构域蛋白将Cdc48募集到叶绿体中。

Nat. Plants (2024). https://doi.org/10.1038/s41477-024-01769-xDownload citationReceived: 09 August 2023Accepted: 20 July 2024Published: 19 August 2024DOI: https://doi.org/10.1038/s41477-024-01769-xShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

《自然植物》（2024）。https://doi.org/10.1038/s41477-024-01769-xDownload引文接收日期：2023年8月9日接收日期：2024年7月20日发布日期：2024年8月19日OI：https://doi.org/10.1038/s41477-024-01769-xShare本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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