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. 2020 Dec 22;117(51):32739-32749.
doi: 10.1073/pnas.2014294117. Epub 2020 Dec 3.

Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

Silvia Ramundo  1   2 , Yukari Asakura  3 , Patrice A Salomé  4 , Daniela Strenkert  4 , Morgane Boone  1   2 , Luke C M Mackinder  5 , Kazuaki Takafuji  6 , Emine Dinc  7   8 , Michèle Rahire  7   8 , Michèle Crèvecoeur  7   8 , Leonardo Magneschi  9 , Olivier Schaad  10 , Michael Hippler  9   11 , Martin C Jonikas  12   2 , Sabeeha Merchant  4 , Masato Nakai  13 , Jean-David Rochaix  14   8 , Peter Walter  15   2
Affiliations

Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

Silvia Ramundo et al. Proc Natl Acad Sci U S A. .

Abstract

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.

Keywords: Chlamydomonas reinhardtii; chloroplast gene targeting; chloroplast protein import; gene coexpression.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Coexpression patterns of Chlamydomonas genes encoding components of the plastid translocon. (A) Correlation matrix for Chlamydomonas genes encoding subunits of the 26S proteasome (listed in Dataset S1). (B) Correlation matrix for Chlamydomonas genes encoding components of the chloroplast translocon identified through BLAST analysis (listed in Dataset S1). (C) Distribution of PCCs for all gene pairs encoding core and regulatory particles of the 26S proteasome (red line) and gene pairs for the chloroplast translocon components, together (magenta line) or as a function of their subgroup, cpA (green line) and cpB (blue line). PCC distribution for all gene pairs in the genome (black line) is shown to indicate the absence of correlation (i.e., negative control). Statistics are available in Dataset S2.
Fig. 2.
Fig. 2.
Coexpression patterns of known and uncharacterized chloroplast translocon components in Chlamydomonas. (A) Correlation matrix of chloroplast translocon genes from subgroups cp1 (comprising most of cpA and cpB genes shown in Fig. 1A), cp2 (containing genes identified during both Tic20 and Tic214 coimmunoprecipitations (reciprocal co-IP) and listed in SI Appendix, Table S3), and cp3 (comprising genes only coexpressed with TIC20 and listed in SI Appendix, Table S4). (B) Venn diagram related to gene subgroups shown in A. (C) PCC distributions for genes belonging to subgroups cp1 (red line), cp2 (magenta line), and cp3 (purple line) and for all gene pairs in the genome (black line) used as negative control. Statistics are available in Dataset S2.
Fig. 3.
Fig. 3.
Coexpression profiles of chloroplast-encoded and nucleus-encoded translocon components over the course of a diurnal cycle in Chlamydomonas. (A) tic214 (black) and orf2971 (light blue) and coexpressed nucleus-encoded translocon components (magenta). (B) tic214 (black), orf528 (blue), and coexpressed plastid-encoded RNA polymerase genes (PEP) (dark red). (C) tic214 (black), psaB (green), and rps12 (brown). Gene expression data are available in Dataset S6.
Fig. 4.
Fig. 4.
Tic20 is part of a stable chloroplast translocon supercomplex in Chlamydomonas. (A) 2D BN/SDS-PAGE separation of the purified Tic20-YFP-FLAG3x fraction (in the presence of digitonin) followed by silver staining. Proteins identified by mass-spectrometric analysis are indicated. (B) Proteins were separated as in A and analyzed by immunoblotting with the indicated antibodies. (C) Isolated chloroplasts from wild-type cells (WT) were solubilized with digitonin and analyzed as in A. (D) Purification of the Tic20-YFP-FLAG3x containing supercomplex was carried out after solubilization with digitonin, DDM, or Triton X-100. The purified fractions (eluate) together with 1% of the total lysate (input) were analyzed by immunoblots with the indicated antibodies. Tic214* in A–D denotes the 110-kDa protein band detected by the Tic214 antibody.
Fig. 5.
Fig. 5.
In vitro import assay of chloroplast preproteins and purification of translocation intermediates in Chlamydomonas. (A) Schematic representation of the two chloroplast preproteins used for in vitro import assays and the purification of translocation intermediates. (B) Chloroplast preproteins were incubated with intact chloroplasts isolated from Chlamydomonas in the presence of the indicated concentrations of ATP. Chloroplasts were reisolated, washed, and directly analyzed (“Tot”) or fractionated into soluble fraction containing stroma (“Sup”). (C) Same assay as in B, except that chloroplasts were treated with thermolysin after protein import. In B, a band corresponding to the mature form of RbcS2 is detected in the absence of ATP and recovered in the Sup fraction. This band does not represent fully translocated protein but is most likely formed or released during the freeze–thaw cycles used for chloroplast lysis. This notion was verified in C with thermolysin treatment, which shows that there is no fully translocated mature RbcS2 in the absence of ATP. (D) Outline of the method used to isolate chloroplast intermediate translocation complexes. After import, washed chloroplasts were solubilized with digitonin, and translocation intermediates were purified with IgG-Sepharose and eluted by TEV protease cleavage. Mock purification was carried out without the addition of preproteins. IE, OE, inner, outer envelope membrane, respectively. (E) Purified fractions shown in D were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies. Tic214* denotes the 110-kDa protein band detected by the Tic214 antibody.
Fig. 6.
Fig. 6.
Conditional depletion of Tic214 inhibits cell growth. (A) Schematic illustration of selective Vit-mediated Tic214 depletion in Y14 cells. The Nac2 protein is translated in the cytosol and imported into the chloroplast, where it is required for expression of the chimeric psbD:tic214 gene. Accumulation of the Nac2 protein and, in turn, of Tic214 is down-regulated upon the addition of thiamine and vitamin B12 (“Vit”) to the growth media. (B) Growth curve of Y14 and the A31 control strains in TAP without or with Vit (20 μg/L vitamin B12 and 200 μM thiamine HCl) under standard light conditions (∼60 μmol m−2 s−1 irradiance). (C) Growth of A31, Y14, REP112, and DCH16 strains was tested by spotting cells on plates containing acetate (TAP) with and without Vit and in TAP supplied with 100 μg/mL spectinomycin (Spec) on which only chloroplast transformants can survive. The chloroplast gene controlled by Nac2 in each strain is indicated at the top. A31 is used as a control since no chloroplast transcript is under the control of the Vit-mediated Nac2 expression in this strain. Expression of the chimeric psbD:tic214 is repressed by Vit in Y14 and expression of the endogenous psbD is repressed by Vit in REP112, whereas the chimeric psbD:clpP1 is repressed by Vit in DCH16 (57, 58). (D) Immunoblot analysis of Tic214 and Vipp2 in Y14 and A31 treated with Vit for the indicated times. The three asterisks indicate a potential SDS-resistant Vipp2 aggregate. Tic214* denotes the 110-kDa protein band detected by the Tic214 antibody. α-Tubulin was used as a loading control. (E) Electron microscopy of Y14 cells supplemented with Vit for 96 h (lower row, “Tic214 OFF”). The control cells without Vit treatment (“Tic214 ON”) are shown in the upper row. (Scale bar, 2 µm.) Intracellular compartments are labeled as follows: Cp, chloroplast; Tk, thylakoids; E, eyespot; SG, starch granules; V, vacuole; N, nucleus.
Fig. 7.
Fig. 7.
Detection of chloroplast precursor proteins upon Tic214 depletion. (A) Schematic representation of the 44 proteins (listed in Dataset S9) for which one or more peptide covering the putative chloroplast transit peptide (cTP) could be detected, selectively upon Tic214 depletion. The green bar indicates the length of the cTP (i.e., number of amino acids) as predicted by ChloroP (69). The circles indicate the first amino acid position of the most N-terminal MS peptide detected in the presence (gray) or absence (magenta) of Tic214. The black asterisks indicate those cases when peptides containing precursor sequences were observed only according to the cTP length predicted by PredAlgo (52). The red and light blue asterisks indicate proteins that have an acetylation on their N-terminal methionine and are ubiquitylated upon Tic214 repression, respectively. (B) Pie chart summarizing the functional classification of the proteins shown in A. Metabolism (n = 21); protein folding/protein degradation (n = 9); ribosome biogenesis/translation (n = 6); photosynthesis (n = 4); other functions or unknown (n = 4). (C) The location and length of peptides spanning the cTP are depicted for some of the proteins shown in A. The light and dark green bars indicate the cTP length as predicted by ChloroP and PredAlgo, respectively. The magenta bars indicate the length of each MS peptide, selectively detected upon Tic214 depletion.
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