V-ATPase复合物组分RNAseK是溶酶体水解酶递送和自噬体降解所必需的-动脉网

The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation

Nature 等信源发布 2024-09-05 10:34

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AbstractAutophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation.

摘要自噬是溶酶体降解胞质成分所需的精心策划的过程。最后的降解步骤对于清除自噬货物和回收大分子至关重要。。

Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK.

对RNAseK敲除细胞的分析表明，虽然自噬体成熟是完整的，但货物降解被严重破坏。重要的是，在不存在RNAseK的情况下，溶酶体蛋白酶活性和酸化保持完整，表明对自溶酶体降解具有特异性。溶酶体级分的分析表明，在不存在RNAseK的情况下，一部分水解酶的水平降低。

Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation..

其中，PLD3的敲低导致自噬体清除缺陷。此外，RNAseK耗尽细胞的溶酶体部分表现出ESCRT-III复合物成分VPS4a的积累，这是PLD3溶酶体靶向所必需的。总而言之，在这里我们确定了有效的自溶酶体降解所需的溶酶体水解酶递送途径。。

IntroductionLysosomes are acidic organelles containing over 60 hydrolases making them major proteolytic compartments in mammalian cells1. Precursors of lysosomal hydrolases are synthesised in the ER and subsequently transported to lysosomes where they are activated2. The luminal acidic environment of lysosomes ensures a contained activity of the hydrolases.

引言溶酶体是含有60多种水解酶的酸性细胞器，使其成为哺乳动物细胞中的主要蛋白水解区室1。溶酶体水解酶的前体在ER中合成，随后转运至溶酶体，在那里它们被激活2。溶酶体的腔酸性环境确保了水解酶的活性。

Delivery of cargo to lysosomes can occur via multiple mechanisms including the endocytic pathway and autophagy. One form of autophagy known as macroautophagy delivers cellular cargo via the formation of double-membrane vesicles, called autophagosomes, which can readily fuse with the lysosomes to form autolysosomes3,4.Autolysosome degradation is vital for the completion of macroautophagy (hereafter referred to as autophagy).

货物向溶酶体的递送可以通过多种机制发生，包括内吞途径和自噬。一种称为巨自噬的自噬形式通过形成称为自噬体的双膜囊泡来传递细胞货物，自噬体可以很容易地与溶酶体融合形成自溶酶3,4。自溶酶体降解对于完成巨自噬至关重要（以下称为自噬）。

Lysosomal enzymes selectively degrade the inner autophagosomal membrane (IAM), while the outer autophagosomal membrane (OAM) remains intact5. IAM disintegration is crucial to expose the autophagosome lumen and subsequent digestion of engulfed cargo, whereas OAM protection is important for the containment of hydrolases within autolysosomes.

溶酶体酶选择性降解内部自噬体膜（IAM），而外部自噬体膜（OAM）保持完整5。IAM崩解对于暴露自噬体腔和随后消化被吞噬的货物至关重要，而OAM保护对于自溶酶体内水解酶的控制很重要。

Immunofluorescence analyses of autolysosomes revealed a formation of a transient acidified ring between the IAM and OAM, suggesting the separation of the two membranes5. In yeast, the integral vacuolar membrane protein Atg15/Cvt17 has been identified as an essential phospholipase required for autophagy and the disintegration of autophagic bodies within the vacuole6.

自体溶酶体的免疫荧光分析显示，在IAM和OAM之间形成了一个瞬时酸化环，表明两个膜分离5。在酵母中，完整的液泡膜蛋白Atg15/Cvt17已被鉴定为自噬和液泡内自噬体崩解所需的必需磷脂酶6。

The specific enzyme(s) responsible for IAM degradation in mammalian cells have not yet been discovered.PLD3 is a member of the PLD phospholipase family. Unlike PLD1 and PLD2, the phospholipase activity of PLD3 is debatable despite the presence of two HKD domains in the protein7,8. Interestingly, PLD3.

尚未发现负责哺乳动物细胞中IAM降解的特定酶。。与PLD1和PLD2不同，尽管蛋白质7,8中存在两个HKD结构域，但PLD3的磷脂酶活性仍存在争议。有趣的是，PLD3。

Data availability

数据可用性

The RNAseK TiD and GFP-VSP4a pulldown proteomics data generated in this study have been deposited in the ProteomeXchange database under accession code PXD042727. The protein content analyses of lysosome-enriched fractions proteomics data generated in this study have been deposited in the ProteomeXchange database under accession code PXD042079.

本研究中产生的RNAseK TiD和GFP-VSP4a下拉蛋白质组学数据已以登录号PXD042727保存在ProteomeXchange数据库中。本研究中产生的溶酶体富集级分蛋白质组学数据的蛋白质含量分析已以登录号PXD042079保存在ProteomeXchange数据库中。

The CRISPR/Cas9 GECKO screen data generated in this study have been deposited in the SRA database under accession code PRJNA977212. Source data are provided with this paper..

本研究中产生的CRISPR/Cas9壁虎筛选数据已保存在SRA数据库中，登录号为PRJNA977212。本文提供了源数据。。

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Download referencesAcknowledgementsWe thank Kevin Myant (University of Edinburgh) for providing lentiviruses for genome-wide CRISPR screen, Stephen Mitchell (University of Edinburgh) for EM sample preparations and help with visualisation, and Val Brunton (University of Edinburgh) for help with the animal work.

下载参考文献致谢我们感谢Kevin Myant（爱丁堡大学）为全基因组CRISPR筛选提供慢病毒，Stephen Mitchell（爱丁堡大学）为EM样品制备和可视化提供帮助，以及Val Brunton（爱丁堡大学）为动物工作提供帮助。

All illustrations were created with BioRender.com using licences numbers (HG270K9GAM, NR270LAA1A). We are grateful to members of the Gammoh laboratory for discussions and critical reading of the manuscript. N.G. is supported by a Cancer Research UK fellowship (C52370/A21586).Author informationAuthors and AffiliationsCancer Research UK Scotland Centre, Institute of Genetics and Cancer, University of Edinburgh, Crewe Road South, Edinburgh, UKAgata N.

所有插图均使用BioRender.com使用许可证编号（HG270K9GAM，NR270LAA1A）创建。我们感谢Gammoh实验室的成员对手稿的讨论和批判性阅读。N、 G.得到英国癌症研究奖学金（C52370/A21586）的支持。作者信息作者和附属机构英国苏格兰研究中心，爱丁堡大学遗传学和癌症研究所，克鲁南路，爱丁堡，UKAgata N。

Makar, Alina Boraman, Joanne E. Simpson, Jair Marques, Tim Michelberger, Alex von Kriegsheim & Noor GammohInstitute for Biochemistry and Molecular Biology, Medical Faculty, University of Bonn, Bonn, GermanyPeter Mosen & Dominic WinterMRC Human Genetics Unit, Institute of Genetics and Cancer, Crewe Road South, University of Edinburgh, Edinburgh, UKStuart Aitken & Ann P.

Makar，Alina Boraman，Joanne E.Simpson，Jair Marques，Tim Michelberger，Alex von Kriegsheim＆Noor Gammoh波恩大学医学院生物化学与分子生物学研究所，德国波恩大学医学院，德国彼得·莫森和多米尼克·温特MRC人类遗传学研究所，爱丁堡大学克鲁南路遗传学与癌症研究所，英国斯图亚特·艾特肯和安·P。

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PubMed Google ScholarContributionsConceptualisation: A.N.M. and N.G.; methodology: A.N.M., A.B., S.A., P.M., D.W., A.V.K., and N.G.; investigation: A.N.M., A.B., J.E.S., J.M., T.M., A.P.W., P.M., A.V.K., and N.G.; writing—original draft: N.G.; writing—review and editing: all authors; supervision: D.W., A.V.K., and N.G.Corresponding authorCorrespondence to.

PubMed谷歌学术贡献概念：A.N.M.和N.G。；方法学：A.N.M.，A.B.，S.A.，P.M.，D.W.，A.V.K。和N.G。；调查：A.N.M.，A.B.，J.E.S.，J.M.，T.M.，A.P.W.，P.M.，A.V.K。和N.G。；撰写原稿：N.G。；写作评论和编辑：所有作者；监督：D.W.，A.V.K。和N.G.相应的作者。

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Nature Communications thanks Jingyue Jia and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Reprints and permissionsAbout this articleCite this articleMakar, A.N., Boraman, A., Mosen, P. et al. The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation.

转载和许可本文引用本文Makar，A.N.，Boraman，A.，Mosen，P。等人。溶酶体水解酶递送和自噬体降解需要V-ATPase复合物成分RNAseK。

Nat Commun 15, 7743 (2024). https://doi.org/10.1038/s41467-024-52049-3Download citationReceived: 02 May 2023Accepted: 23 August 2024Published: 05 September 2024DOI: https://doi.org/10.1038/s41467-024-52049-3Share 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.

《国家公社》157743（2024）。https://doi.org/10.1038/s41467-024-52049-3Download引文接收日期：2023年5月2日接收日期：2024年8月23日发布日期：2024年9月5日OI：https://doi.org/10.1038/s41467-024-52049-3Share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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