Chloroplast Biogenesis Is Regulated by Direct Action of the Ubiquitin-Proteasome System

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Science  02 Nov 2012:
Vol. 338, Issue 6107, pp. 655-659
DOI: 10.1126/science.1225053


Development of chloroplasts and other plastids depends on the import of thousands of nucleus-encoded proteins from the cytosol. Import is initiated by TOC (translocon at the outer envelope of chloroplasts) complexes in the plastid outer membrane that incorporate multiple, client-specific receptors. Modulation of import is thought to control the plastid’s proteome, developmental fate, and functions. Using forward genetics, we identified Arabidopsis SP1, which encodes a RING-type ubiquitin E3 ligase of the chloroplast outer membrane. The SP1 protein associated with TOC complexes and mediated ubiquitination of TOC components, promoting their degradation. Mutant sp1 plants performed developmental transitions that involve plastid proteome changes inefficiently, indicating a requirement for reorganization of the TOC machinery. Thus, the ubiquitin-proteasome system acts on plastids to control their development.

Chloroplasts belong to a family of plant organelles called plastids, which includes several nonphotosynthetic variants (such as etioplasts in dark-grown seedlings and carotenoid-rich chromoplasts in fruits) (1). A specific feature of the plastid family is the ability to interconvert in response to developmental and environmental cues—for example, during de-etiolation or fruit ripening (1). Such plastid interconversions are linked to reorganization of the organellar proteome (2, 3).

Over 90% of the thousands of proteins in plastids are nucleus-encoded and imported from the cytosol posttranslationally (1). The translocon at the outer envelope of chloroplasts (TOC) recognizes chloroplast pre-proteins and initiates their translocation (46). The TOC machinery comprises the Omp85 (outer membrane protein, 85 kD)–related channel Toc75 and the receptor guanosine triphosphatases Toc34 and Toc159. The receptors protrude into the cytosol, where different isoforms contact pre-proteins with differing specificity: In Arabidopsis, the major isoforms (atToc33 and atToc159) recognize abundant precursors of the photosynthetic apparatus, whereas the minor isoforms (atToc34 and atToc132/atToc120) recognize housekeeping pre-proteins (710). Receptor isoform levels vary developmentally depending on biochemical requirements of the plastids.

Although the main TOC components were identified more than a decade ago, regulatory mechanisms that govern their action are poorly understood. To shed light in this area, we screened an ethyl methanesulfonate–mutagenized population of Arabidopsis for second-site suppressors of the atToc33 knockout mutation, plastid protein import1 (ppi1; which causes chlorosis due to defective protein import) (7), and identified suppressor of ppi1 locus1 (sp1). Double-mutant sp1 ppi1 plants were larger and greener than were ppi1 and exhibited improved chloroplast ultrastructural organization and protein import capacity (Fig. 1). Recovery mediated by sp1 was specific; two other mutations that cause chlorosis were not suppressed (fig. S1). The only other mutation found to be suppressed by sp1 was a hypomorphic allele of the gene encoding Toc75 (toc75-III-3) (11, 12), implying a close functional relationship between SP1 and the TOC apparatus.

Fig. 1

The sp1 mutation suppresses the phenotype of the atToc33 knockout mutation, ppi1. (A) Plants grown on soil for 30 days. (B) Leaf chlorophyll contents of similar 40-day-old plants. (C) Ultrastructure of typical cotyledon chloroplasts in 10-day-old plants grown in vitro. Scale bar, 2 μm. These and other micrographs were used to estimate (D) chloroplast cross-sectional area and (E) thylakoid development. (F) Protein import into isolated chloroplasts was measured by quantifying maturation (mat) of in vitro translated (IVT) Rubisco small subunit precursors (pre). (G) Analysis of chloroplast proteins by SDS–polyacrylamide gel electrophoresis and SYPRO (Invitrogen, Eugene, Oregon) staining, revealing sp1-linked restoration of the three main photosynthetic proteins: Rubisco large (LSU) and small (SSU) subunits; light-harvesting chlorophyll-binding protein (LHCP). All values are means ± SEM (n ≥ 4 experiments or samples).

The SP1 locus (At1g63900) was identified by means of map-based cloning; the original sp1-1 allele carries a splice-junction mutation, causing frame-shifts, whereas two insertional mutants (sp1-2 and sp1-3) also lack the native SP1 transcript and are phenotypically similar (fig. S2). SP1 is a putative C3HC4-type really interesting new gene (RING) ubiquitin E3 ligase (13, 14). Such E3s perform a crucial role in the ubiquitin-proteasome system (UPS), along with E1 and E2 enzymes and the 26S proteasome. The UPS is a central proteolytic system in eukaryotes with numerous components, accounting in Arabidopsis for ~6% of the proteome (15). The E1, E2, and E3 enzymes cooperate to attach ubiquitin to target proteins, which are then typically degraded by the proteasome. Targets are identified primarily by the E3s, of which there are many (~90% of 1600 UPS components in Arabidopsis are E3s), enabling specific recognition (and regulation) of numerous, functionally diverse substrates (15). Unimported plastid pre-proteins in the cytosol are UPS substrates (16, 17), but whether the plastid itself is a target is not clear. Overexpression of SP1 accentuated the phenotypes of TOC mutants, supporting the notion that it regulates the import machinery (fig. S3).

The SP1 protein has two predicted transmembrane spans (Fig. 2A). Translational fusions to yellow fluorescent protein (YFP) indicated localization to the chloroplast envelope that was dependent on these transmembrane domains (Fig. 2B). In isolated chloroplasts, SP1 was resistant to alkaline extraction and partially sensitive to applied proteases (Fig. 2, C and D, and fig. S4), indicating that it is an integral outer-membrane protein with an intermembrane-space domain, and that the RING domain is cytosolically oriented and accessible to UPS components. Localization of SP1 to chloroplasts (a major source of reactive oxygen species) may explain why SP1 is linked to programmed cell death (18).

Fig. 2

SP1 is located in the chloroplast outer-envelope membrane with its RING domain facing the cytosol. (A) SP1 protein map showing transmembrane (TMD), intermembrane space (SP1ims), cytosolic (SP1cyt), and RING finger (RNF) domains. (B) Localization of SP1-YFP to chloroplast envelopes (top) depended on the transmembrane domains, as revealed by a double-deletion mutant (bottom). Scale bar, 10 μm. (C) Radiolabeled SP1 in isolated chloroplasts was located in the membrane pellet (P) fraction after high-pH washing, in contrast with imported mature SSU which was in the soluble (S) fraction. Endogenous markers partitioned as expected (Coomassie stain; bottom). (D) Radiolabeled SP1 and C-terminally tagged SP1–hemagglutinin (HA)–FLAG were imported into chloroplasts before their treatment with thermolysin (Th), trypsin (Tryp), thermolysin plus Triton X-100 (Th/TX) (Fisher Scientific, Fair Lawn, New Jersey), or buffer lacking protease (Mock). Phosphor-imaging revealed protease sensitivity and protected fragments (of sizes not influenced by the C-terminal tag) consistent with outer membrane localization and the topology shown in (A). Immunoblot analysis of three endogenous markers confirmed efficacy of the treatments.

Two SP1 homologs exist in Arabidopsis: SP1-Like1 (SPL1) and SPL2. They share topological similarity and considerable sequence identity with SP1, and both were located in the chloroplast envelope (fig. S5). However, overexpression of SP1’s closest relative, SPL1, did not complement sp1 (fig. S6), suggesting that SP1 and its homologs have distinct clients. Also related to SP1 is the human mitochondrial outer-membrane protein MULAN/MAPL, which controls mitochondrial dynamics (1922).

In planta, activity of SP1 depended on the presence of a functional RING domain (fig. S7, A to D), which in E3s is required for E2 recruitment (14, 15). Purified SP1 had self-ubiquitination activity (13), typical for E3s, which was similarly dependent on RING functionality (fig. S7, E and F). Polyubiquitinated SP1 was also detected in plants, in amounts proportional to RING integrity (fig. S8A). Such auto-ubiquitination implies that SP1 itself is subject to UPS control, as E3s frequently are (14). Accordingly, cellular SP1 protein levels were elevated upon treatment with the proteasome inhibitor MG132 (fig. S8B) (17, 23).

Phenocopy of sp1-mediated suppression was observed when 26S proteasome mutants (24) were crossed to ppi1 or toc75-III-3 (fig. S9), suggesting that the UPS indeed controls chloroplast development. We thus set about identifying the target (or targets) of SP1 E3 activity. All tested TOC proteins were deficient in ppi1 relative to wild type but substantially recovered in sp1 ppi1 (Fig. 3A); other envelope proteins [Tic110, Tic40, OEP80, and SFR2 (25)] were largely unaffected by sp1. These changes were not attributable to pretranslational events because TOC transcript levels were comparable in the different genotypes (fig. S10A). Similar TOC protein abundance recovery was apparent in sp1 toc75-III-3 (fig. S10B). TOC protein levels were also elevated in the visibly normal sp1 single mutant, arguing against the possibility that the protein changes in sp1 ppi1 and sp1 toc75-III-3 were a consequence (rather than a cause) of the phenotypic recovery. Moreover, SP1 overexpression preferentially depleted TOC proteins (Fig. 3A and fig. S10C); effects on other envelope proteins in the ppi1 background were likely indirect consequences of general phenotype severity (Fig. 3A and fig. S3).

Fig. 3

SP1 associates with TOC complexes and targets TOC components for UPS-mediated degradation via ubiquitination. (A) Immunoblot analysis of total leaf protein from different genotypes, including SP1 overexpressors (OX). Plasma membrane H+-ATPase, PMA2, acted as a loading control. Bars show means ± SEM (n = 4 to 6 experiments). (B) Coimmunoprecipitation (IP) of TOC components with HA-tagged SP1 from protoplast extracts. Cells were transfected with an SP1-HA construct or empty vector (v). (C) In vitro pull-down of radiolabeled TOC components [or domains (25)] with GST-SP1 baits. (D) In vitro ubiquitination of radiolabeled TOC components (but not atToc159G) by recombinant GST-SP1flex. Asterisks indicate a nonspecific 48-kD band seen in all translations. Mono-ubiquitinated atToc159G would be expected to migrate near the 50 kD marker. (E) Ubiquitination of atToc33 as in (D) using free and HA-tagged ubiquitin (8.5 and 9.4 kD, respectively). (F) Immunoprecipitation of TOC components with FLAG-tagged ubiquitin from transfected protoplasts. (G) Immunoprecipitation under denaturing conditions of FLAG-ubiquitin with atToc33; a control IP used excess anti-atTic110. Immunoglobulin G heavy chain (hc) is shown. Ubiquitinated species (Ub) are indicated [(D) to (G)].

Consistent with the notion that TOC proteins are targeted for UPS-mediated degradation by SP1, all three principal TOC components coimmunoprecipitated with epitope-tagged SP1 from plant extracts (Fig. 3B). In vitro pull-down experiments revealed SP1 interactions with Toc75 and all tested TOC receptors (Fig. 3C), which is not unusual because E3s often have diverse substrates (14, 15). These interactions were mediated primarily by the SP1 intermembrane-space domain and the membrane/intermembrane-space domains of the receptors.

In vitro ubiquitination assays using radiolabeled TOC proteins, purified SP1, and UPS components revealed high-molecular-weight species indicative of TOC ubiquitination (Fig. 3D). Some ubiquitination occurred in the absence of E3 [presumably mediated by E2 alone (26)], but for each TOC substrate, the extent of ubiquitination was enhanced in the presence of SP1. The identity of mono-ubiquitinated atToc33 was confirmed by a size shift upon utilization of different forms of ubiquitin (Fig. 3E).

In an in vivo assay for ubiquitination, the three main TOC components all co-immunoprecipitated with epitope-tagged ubiquitin in amounts proportional to the expression of SP1 (in sp1, amounts were less than in wild type; in an SP1 overexpressor, amounts were more) (Fig. 3F). Moreover, modified forms of atToc159 and atToc33 were apparent in the precipitates, which likely correspond to ubiquitinated species. Absence of clearly ubiquitinated forms of Toc75 may indicate that it is less readily ubiquitinated than the receptors in vivo, or more readily de-ubiquitinated (27). Regardless, its association with other ubiquitinated TOC proteins may be sufficient to promote its turnover via the UPS (28). In a reciprocal experiment (performed under denaturing conditions to disrupt noncovalent protein-protein associations; see atToc159 control), high-molecular-weight ubiquitin smears were apparent in atToc33 immunoprecipitates (Fig. 3G). Abundance of polyubiquitinated atToc33 was controlled by proteasomal activity, as revealed by MG132 treatment. Thus, TOC components are indeed ubiquitinated in vivo, which controls their turnover. Genetic suppression by sp1 is likely due to the stabilization of TOC components (such as atToc75-III and atToc34).

Our data imply a role for SP1 in the reorganization of the TOC machinery (fig. S11) and a mechanism for the regulation of plastid biogenesis. This might be important during developmental phases in which plastids convert from one form to another through organellar proteome changes (13). For example, during fruit ripening in crops such as tomato and citrus, chloroplasts differentiate into chromoplasts, which accumulate carotenoid pigments of dietary importance (3). In Arabidopsis, when etiolated seedlings are exposed to light, heterotrophic etioplasts rapidly differentiate into chloroplasts (29). This is essential for initiation of photoautotrophic growth after seed germination beneath the soil. In accordance with the hypothesis, sp1 single mutants de-etiolated inefficiently, displaying reduced survival rates linked to delayed organellar differentiation (Fig. 4, A to E), reduced accumulation of photosynthetic proteins, and imbalances in TOC receptor levels (fig. S12). At the other end of the life cycle, chloroplasts transform into gerontoplasts as catabolic enzymes accumulate to recover resources from the organelles of senescent leaves for use elsewhere in the plant. This response is characterized by declining photosynthetic performance and can be induced prematurely by dark treatment (30). The sp1 mutation also attenuated this transition (Fig. 4, F and G), whereas SP1 overexpression enhanced both senescence and de-etiolation (Fig. 4), presumably because of the hastening of organellar proteome changes.

Fig. 4

SP1 is important for developmental processes that require reorganization of the plastid proteome. (A to E) De-etiolation of seedlings grown in darkness for 6 [(A) to (D)] or 5 (E) days, upon transferral to continuous light. [(A) and (B)] Cotyledons of typical plants, and survival rates, after two days of illumination. [(C) and (D)] Ultrastructure of typical cotyledon plastids after 0, 6, and 24 hours of illumination, and proportion of plastids at each of three progressively more advanced developmental stages (25) after 6 hours of illumination. Scale bar, 2 μm. (E) Chlorophyll contents after 16 hours of illumination. (F and G) Senescence of leaves induced by covering with aluminum foil. (F) Typical control (uncovered) and senescent (covered) leaves. (G) Photochemical efficiency of photosystem II (Fv/Fm) was measured to estimate the extent of senescence. All values are means ± SEM (n = 3 to 9 experiments or samples).

Identification of plastids as targets of UPS activity extends the known field of influence of this remarkably pervasive eukaryotic regulatory network. Although its direct action may be limited to cytosolically exposed proteins of the plastid’s outer membrane, this may orchestrate wholesale internal changes through reorganization of the protein import machinery.

Supplementary Materials

Materials and Methods

Figs. S1 to S12

Table S1

References (3163)

References and Notes

  1. Materials and methods and further information are available as supplementary materials on Science Online.
  2. Acknowledgments: We thank M. Rashbrooke for assistance with initial phenotype analyses and rough mapping of sp1; U. Ranganathan for her contribution to the analysis of SPL1; R. Patel and R. Berkeley for excellent technical assistance; N. Allcock and S. Hyman for electron microscopy (EM) carried out within the EM Laboratory, University of Leicester; C. Dean and R. Trösch for helpful comments on the manuscript; U. Flores-Perez for antigen preparation (atToc132 and atToc34); M. Boutry (PMA2), N.E. Hoffman (LHCP), K. Inoue (OEP80), F. Kessler (atToc159), and G. Thorlby (SFR2) for antibodies; C.E. Stebbins for the AtUBC8 clone; and the Salk Institute Genomic Analysis Laboratory (SIGnAL) and the Nottingham Arabidopsis Stock Centre (NASC) for the sp1-2 and sp1-3 alleles. This study was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC; BB/D016541/1 and BB/H008039/1) to P.J., by the Royal Society Rosenheim Research Fellowship to P.J., and by a Royal Society International Incoming Fellowship to W.H. This work is the subject of patent application number GB 1216090.9, which covers the role of the ubiquitin-proteasome system in the control of plastid development. The data are presented in the manuscript and in the supplementary materials.
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