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. 2011 Nov;23(11):3911-28.
doi: 10.1105/tpc.111.092882. Epub 2011 Nov 29.

Plastid proteome assembly without Toc159: photosynthetic protein import and accumulation of N-acetylated plastid precursor proteins

Sylvain Bischof  1 , Katja Baerenfaller, Thomas Wildhaber, Raphael Troesch, Pierre-Alexandre Vidi, Bernd Roschitzki, Matthias Hirsch-Hoffmann, Lars Hennig, Felix Kessler, Wilhelm Gruissem, Sacha Baginsky
Affiliations

Plastid proteome assembly without Toc159: photosynthetic protein import and accumulation of N-acetylated plastid precursor proteins

Sylvain Bischof et al. Plant Cell. 2011 Nov.

Abstract

Import of nuclear-encoded precursor proteins from the cytosol is an essential step in chloroplast biogenesis that is mediated by protein translocon complexes at the inner and outer envelope membrane (TOC). Toc159 is thought to be the main receptor for photosynthetic proteins, but lacking a large-scale systems approach, this hypothesis has only been tested for a handful of photosynthetic and nonphotosynthetic proteins. To assess Toc159 precursor specificity, we quantitatively analyzed the accumulation of plastid proteins in two mutant lines deficient in this receptor. Parallel genome-wide transcript profiling allowed us to discern the consequences of impaired protein import from systemic transcriptional responses that contribute to the loss of photosynthetic capacity. On this basis, we defined putative Toc159-independent and Toc159-dependent precursor proteins. Many photosynthetic proteins accumulate in Toc159-deficient plastids, and, surprisingly, several distinct metabolic pathways are negatively affected by Toc159 depletion. Lack of Toc159 furthermore affects several proteins that accumulate as unprocessed N-acetylated precursor proteins outside of plastids. Together, our data show an unexpected client protein promiscuity of Toc159 that requires a far more differentiated view of Toc159 receptor function and regulation of plastid protein import, in which cytosolic Met removal followed by N-terminal acetylation of precursors emerges as an additional regulatory step.

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Figures

Figure 1.
Figure 1.
Quantitative Proteome Profiling of ppi2, Toc159cs, Wild-Type, and wtS Leaves. (A) to (D) Phenotypes of 35-d-old plants used for large-scale proteome profiling grown in short-day conditions (8 h light/16 h dark) on soil or on half-strength Murashige and Skoog medium supplemented with 100 mM Suc for ppi2 and wtS. ppi2 (A), Toc159cs (B), wild type (C), and wtS (D). Bars = 5 mm. (E) Protein immunoblotting using antibodies directed against Toc159 confirming the lack of Toc159 in ppi2 and Toc159cs plants. Coomassie blue staining shows an equivalent protein loading for each protein extract separated by SDS-PAGE. wt, wild type. (F) Proteins identified by MS/MS in ppi2, Toc159cs, wild-type, and wtS leaves. Total proteins were extracted from leaves, separated by gel electrophoresis, digested with trypsin, and analyzed by liquid chromatography MS/MS using a linear trap quadrupole Fourier transform–ion cyclotron resonance mass spectrometer.
Figure 2.
Figure 2.
Quantitative Accumulation of Plastid Proteins Identified in Leaves. (A) Venn diagram of the plastid proteins identified in leaves by MS/MS. Plastid localization is based on a chloroplast proteome reference table with 1155 proteins. wt, wild type. (B) Differential accumulation of nuclear-encoded plastid proteins in wild-type and Toc159-deficient leaves. Numbers have been rounded to one decimal. (C) Metabolic annotation of the nuclear-encoded genes destined to the plastid based on their distribution in MapMan bins. Categories indicated with an asterisk are significantly overrepresented, showing P values < 1e-05 in Fisher’s exact test. CHO, carbohydrate; OPP, oxidative pentose phosphate.
Figure 3.
Figure 3.
Transcriptional Response in ppi2 Compared with wtS. (A) Expression data for 24,639 genes of ppi2 compared with wtS grown on Suc (all genes) and for the subset of 1046 nuclear-encoded plastid proteins. The percentage of downregulated genes in ppi2 highly increases when only nuclear-encoded genes destined to the plastid are taken into account. (B) Metabolic annotation of the nuclear-encoded genes destined to the plastid based on their distribution in MapMan bins. Categories indicated with an asterisk are significantly overrepresented, showing P values < 1e-05 in Fisher’s exact test. (C) Expression data for the genes for which the proteins were found significantly upregulated in wild type (wt)/wtS, unchanged, or significantly upregulated in toc159cs/wtS in the proteomics data. CHO, carbohydrate; OPP, oxidative pentose phosphate.
Figure 4.
Figure 4.
Peptide Detection Incidence and Transit Peptide Cleavage in Leaves. (A) Peptide minimal position of plastid proteins identified in leaves. The positions of the most N-terminal detected peptide of each identified protein were grouped into bins. The reduced detection of peptides at the N terminus of plastid proteins supports the removal of transit peptides after import in the wild type and in Toc159-deficient plants. The number of proteins for each blot (wild type, wtS, Toc159cs, and ppi2) is 763, 771, 645, and 659. (B) Peptide minimal position of nonplastid proteins identified in leaves indicates that the N terminus of most nonplastid proteins is not cleaved and readily detectable by MS. The number of proteins for each blot (wild type, wtS, Toc159cs, and ppi2) is 1936, 2051, 2427, and 2444.
Figure 5.
Figure 5.
Met Removal and N-Terminal Acetylation in Leaves. (A) N-acetylation sites of plastid proteins identified by MS/MS. N-acetylated proteins were grouped according to the position of their N-acetylation site. N-acetylation sites of plastid proteins identified at positions 30 to 80 reflect the correct cleavage and processing of transit peptides after import in wild-type and in Toc159-deficient plants. The increased percentage of N-acetylated plastid proteins at positions 1 to 10 in Toc159-deficient leaves suggests their accumulation outside plastids as a consequence of partially impaired import. The number of proteins for each blot (wild type, wtS, Toc159cs, and ppi2) is 42, 48, 37, and 33. (B) Distribution of N-acetylation sites of nonplastid proteins indicates that most of these undergo Met removal and N-acetylation in the cytosol. The number of proteins for each blot (wild type, wtS, Toc159cs, and ppi2) is 121, 140, 191, and 168. (C) Schematic representation of the cytosolic two-step Met removal/acetylation pathway. (1) N-terminal start Met is usually removed from newly synthesized precursor proteins containing a transit peptide (TP) if the second residue is small and uncharged. (2) Addition of N-acetylation at the second residue by N-acetyltransferases. (D) Sequence alignment of N-acetylation sites (Ac) of all plastid proteins N-acetylated at position 2 in wild-type and in Toc159-deficient plants.
Figure 6.
Figure 6.
Sequence Context around N-Acetylation Sites of Plastid Proteins Identified in Leaves. Most plastid proteins are N-acetylated (Ac) on Val or Ala. The similar sequence context in wild-type and in Toc159-deficient plants indicates that precursor processing of imported proteins is also functional in the mutant lines. N-acetylation sites ranging between positions 25 and 90 were used for the alignment.
Figure 7.
Figure 7.
Comparison of N-acetylation Sites Identified in Isolated Plastids and Leaves. (A) Similar distribution patterns of N-acetylation sites identified in isolated the wild-type and ppi2 plastids indicate that plastid proteins were imported and processed correctly. The number of proteins for each blot (the wild type and ppi2) is 68 and 42. (B) Similar distribution patterns of N-acetylation sites between isolated wild-type chloroplasts and wild-type leaves. The number of proteins for each blot (leaves and plastids) is 42 and 68. (c) Comparison of the N-acetylation sites between isolated ppi2 chloroplasts and ppi2 leaves. A major difference (*) is observed at positions 1-10 suggesting the accumulation outside plastids of unprocessed precursor proteins when import is partially impaired in ppi2. The number of proteins for each blot (leaves and plastids) is 33 and 42.
Figure 8.
Figure 8.
Transit Peptide Visualization and Extracted Ion Chromatogram Quantification for Phosphoglycerate Kinase1 Peptides Identified in Leaves and Isolated Plastids. (A) Visualization of peptide detection along the protein. Several spectra matched to the transit peptide region (indicated as TP; 75 amino acids predicted length) in Toc159-deficient (toc159) leaves. By contrast, we could not detect any spectra matching the predicted TP region in wild-type leaves or in isolated ppi2 or wild-type plastids, although the coverage of Phosphoglycerate Kinase1 (PGK1) detection is similar between the different samples (cf. peptide color scheme and peptide localization between the different samples; the darker the shading, the more spectra matching to the indicated peptide were observed). (B) Extracted exact mass ion chromatogram for the spectra matching to the transit peptide region from leaves (top panel; marked with an asterisk) and corresponding samples from isolated plastids. In three samples from isolated plastids, an ion matching the exact mass of the PGK1 transit peptide identification (asterisk) was detected at a shifted retention time of ~8 min (bottom panels). MS/MS information on these ions revealed that they are clearly different from the peptide eluting at 48 min (top panel, precursor peptide). The upper peptide represents the peptide matching to the PGK1 transit peptide, the three ions below produced a poor-quality MS/MS spectrum that could not be assigned to any peptide (it was assigned to format dehydrogenase with a score below the threshold). L, leaves; P, plastids. [See online article for color version of this figure.]
Figure 9.
Figure 9.
Toc159-Independent Import of 35S:PGL35 in White Toc159cs Leaves. (A) Epidermal cells of wild-type and white Toc159cs leaves transformed by biolistic transformation. GFP fused to the transit peptide of PGL35 is imported successfully into wild-type and Toc159cs plastids. Maximal projections of confocal images are shown. (B) Import into plastids in wild-type and white Toc159cs leaves of GFP fused to the transit peptide of ribulose-1,5-bis-phosphate carboxylase/oxygenase small subunit (pSSU). Bars = 10 μm.
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