Research Article

Ubiquitin-dependent chloroplast-associated protein degradation in plants

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Science  22 Feb 2019:
Vol. 363, Issue 6429, eaav4467
DOI: 10.1126/science.aav4467

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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


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.


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.


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.


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.


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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