Ubiquitin-dependent chloroplast-associated protein degradation in plants
Chloroplast-associated protein degradation
Protein degradation is vital for cellular functions, and it operates selectively with distinct mechanisms in different organelles. Some organellar proteins are targeted by the ubiquitin-proteasome system (UPS)—a major proteolytic network in the eukaryotic cytosol. In such cases, the organelle membrane presents a substantial barrier to protein degradation. Working in the model plant Arabidopsis, Ling et al. identified mechanisms underlying the UPS-mediated degradation of proteins in the outer membrane of chloroplasts (the organelles responsible for photosynthesis). They identified an Omp85-type β-barrel outer membrane channel and a cytosolic AAA+ chaperone that fulfill conductance and motor functions in the retrotranslocation of target proteins from chloroplasts. This process thus enabled outer membrane protein processing by the cytosolic proteasome. Such chloroplast-associated protein degradation was initiated by ubiquitination of the targets by the chloroplast-localized E3 ubiquitin ligase SP1.
Science, this issue p. eaav4467
Structured Abstract
INTRODUCTION
Chloroplasts are plant organelles responsible for the bulk of terrestrial photosynthetic primary production. They evolved via endosymbiosis from a cyanobacterial organism more than a billion years ago. The biogenesis and operation of chloroplasts depends on the assembly and homeostasis of thousands of nucleus-encoded proteins, which together constitute a large part of the organellar proteome. These proteins are imported by multiprotein translocases in each of the chloroplast envelope membranes after translation in the cytosol. Chloroplast proteins are subject to proteolytic regulation, which plays vital roles in maintaining normal organellar functions and in delivering responses to developmental and environmental cues. Turnover of internal chloroplast proteins is controlled by proteases inherited from the organelle’s prokaryotic ancestor, but mechanisms underlying the degradation of chloroplast outer envelope membrane (OEM) proteins are poorly defined.
RATIONALE
We previously showed that components of the protein import translocases in the OEM (so-called TOC proteins) are ubiquitinated by the OEM-localized ubiquitin E3 ligase SP1 and subsequently degraded by the cytosolic 26S proteasome. Inherent in this process is a need to extricate the target proteins from the chloroplast membrane (they are integral membrane proteins), and to achieve this there must exist mechanisms to overcome the physical and thermodynamic barriers to extraction. This implies that additional factors are involved in OEM protein degradation, and we sought to identify these by applying forward genetics and proteomic analysis in the plant Arabidopsis.
RESULTS
We identified two factors required for the degradation of chloroplast OEM proteins: SP2 and CDC48. The former is an Omp85-type β-barrel channel of prokaryotic origin located in the OEM, and the latter is a conserved eukaryotic AAA+ chaperone located in the cytosol. We observed that inactivation of either component triggers the selective overaccumulation of target proteins, specifically at the chloroplast envelope. We used genetic analyses to demonstrate that SP2 and CDC48 act together in the same proteolytic pathway as the SP1 E3 ligase and physical interaction studies to show that the three components can form a complex at the surface of the chloroplast. Furthermore, by applying complementary in vivo and in vitro assays, we demonstrated that the SP2 and CDC48 proteins cooperate to bring about the extraction (“retrotranslocation”) of ubiquitinated proteins from the OEM. Overall, the data are consistent with a model (see the figure) in which SP2 and CDC48 fulfil conductance and motor functions, respectively, in the retrotranslocation of OEM proteins ubiquitinated by SP1 to enable their proteasomal degradation in the cytosol. These results extend the range of known functions of Omp85 superfamily proteins (which heretofore included bacterial protein secretion, membrane protein biogenesis, and organelle protein import) and of CDC48 (which has a well-characterized role in endoplasmic reticulum–associated protein degradation). The broader importance of this proteolytic mechanism was demonstrated by physiological analyses of plants with altered SP2 activity, which revealed defects in organellar functions, plant development, and viability.
CONCLUSION
Collectively, our results describe a multicomponent system for chloroplast envelope protein removal, dependent on the cytosolic ubiquitin-proteasome system, which is critically important for plant growth. A key part of the system is a protein retrotranslocation mechanism of chimeric prokaryotic-eukaryotic ancestry that operates at the surface of the organelle. We refer to this proteolytic system as chloroplast-associated protein degradation, or CHLORAD.
Chloroplast-associated protein degradation.
CHLORAD is a proteolytic system that selectively removes chloroplast OEM proteins, including TOC components of the chloroplast protein import machinery. The SP1 E3 ligase directs the ubiquitination (Ub) of targets; it has a RING finger (RNF) domain for ubiquitin-conjugating enzyme (E2) recruitment and an intermembrane space (IMS) domain that binds to its targets. The SP2 and CDC48 proteins mediate target retrotranslocation to the cytosol, respectively providing a conduit and driving force for the process. Upon release to the cytosol, targets are degraded by the 26S proteasome (26SP). Additional, as yet unknown factors are shown in gray.
Abstract
Chloroplasts contain thousands of nucleus-encoded proteins that are imported from the cytosol by translocases in the chloroplast envelope membranes. Proteolytic regulation of the translocases is critically important, but little is known about the underlying mechanisms. We applied forward genetics and proteomics in Arabidopsis to identify factors required for chloroplast outer envelope membrane (OEM) protein degradation. We identified SP2, an Omp85-type β-barrel channel of the OEM, and CDC48, a cytosolic AAA+ (ATPase associated with diverse cellular activities) chaperone. Both proteins acted in the same pathway as the ubiquitin E3 ligase SP1, which regulates OEM translocase components. SP2 and CDC48 cooperated to bring about retrotranslocation of ubiquitinated substrates from the OEM (fulfilling conductance and motor functions, respectively), enabling degradation of the substrates by the 26S proteasome in the cytosol. Such chloroplast-associated protein degradation (CHLORAD) is vital for organellar functions and plant development.
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Supplementary Material
Summary
Figs. S1 to S20
Tables S1 and S2
Resources
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Information & Authors
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Published In
Science
Volume 363 | Issue 6429
22 February 2019
22 February 2019
Copyright
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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Submission history
Received: 17 September 2018
Accepted: 15 January 2019
Published in print: 22 February 2019
Acknowledgments
We thank M. Rashbrooke for assistance with initial analyses and rough mapping of sp2; Y. Zeng, J. Bédard, and N. Li for technical assistance; N. Allcock and S. Hyman (University of Leicester EM Laboratory) for electron microscopy; A. R. Bottrill (University of Leicester Protein Nucleic Acid Chemistry Laboratory) for mass spectrometry; L. Dolan for comments on the manuscript; L. J. Sweetlove (Slp1), J. Denecke (calreticulin), and B. Bartel (PEX13) for antibodies; F. Wu for the Toc159 BiFC construct; S. Y. Bednarek for the H6T7-DN-B construct; G. Benvenuto (and Addgene) for the pE3c construct; J. Chory and J. Woodson for ppi2-3 (fts1) seeds; and SIGnAL and NASC for the sp2-4 allele. Funding: This work was supported by grants from the BBSRC (BB/D016541/1, BB/K018442/1, BB/R009333/1, and BB/R016984/1) to R.P.J.; the Royal Society Rosenheim Research Fellowship to R.P.J.; a Department of Plant Sciences DPhil studentship to R.P.J and W.B.; a Gatsby Sainsbury Ph.D. studentship to R.T.; and a Carl Tryggers Stiftelse för Vetenskaplig Forskning fellowship (CTS 11:479) to M.T. Author contributions: Q.L. and W.B. formulated the research plan, designed and performed experiments, and interpreted results. Q.L. carried out the functional analysis of CDC48 and contributed to the preparation of the manuscript. W.B. completed genetic, molecular, physiological, and detailed functional studies of SP2, with assistance from Q.L. R.T. performed the genetic mapping of sp2, prepared samples for whole-genome sequencing, and conducted protein import assays with assistance from T.D.S. M.T. analyzed the whole-genome sequencing data and identified a candidate sp2 locus. P.L. performed the TAP experiment. A.B. conducted the mutant screen and identified the original sp2 alleles. R.P.J. conceived of the study, supervised the work, analyzed the data, and prepared the manuscript. All authors discussed the results and commented on the manuscript. Competing interests: This work is the subject of pending UK patent applications GB 1803833.1, GB 1803834.9, and GB 1815206.6 (the inventor is R.P.J. in each case), which cover the use of CHLORAD to manipulate plastid development in crops. Data and materials availability: Sequencing data used in this study, as well as the reference genome for ppi1, are available at the National Center for Biotechnology Information under Bioproject PRJNA488548. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRoteomics IDEntifications (PRIDE) repository with the identifier PXD010954. All other data are available in the manuscript or the supplementary materials.
Authors
Funding Information
Biotechnology and Biological Sciences Research Council: BB/D016541/1, BB/K018442/1-2, BB/R009333/1
Royal Society: Rosenheim Research Fellowship
Gatsby Charitable Foundation: Sainsbury PhD Studentship
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