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Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.

Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson O, Sun Q, van Wijk KJ - PLoS ONE (2008)

Bottom Line: The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude.Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction.No evidence was found for suggested targeting via the secretory system.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models.

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Quantification of the chloroplast protein homeostasis network including processing, (un)folding, maturation and proteolysis.Relative concentrations of 48 stromal proteins involved in the post-translational protein homeostasis network are displayed with color coding. Abbreviations are as follows: SPP, general stromal processing peptidase; PDF1A,B, methionine deformylases 1A,B; MSRA4,B2, methionine sulfoxide reductases; AP, amino-peptidases; CPN10,20,60, chaperone 10,20 and 60 of the GroEL/ES system; cpHSP70, heat shock protein 70; GrpE, nucleotide exchange factor; HSP90, heat shock protein 90; ClpB3, chaperone B3; cpSRP – chloroplast signal recognition particle subunit 43 and 54, involved in protein targeting components cpSRP43, cpSRP54; cpSecA – ATP-dependent Sec targeting component; cpTIG, a homologue of E. coli trigger factor involved in protein folding at the ribosome; ROC4, protein isomerase with unknown function; ClpP/R/S,T,C,D- subunits of the complete Clp protease system, DegP2 – protease of the Deg family; AtPrep1 - a Zn-protease suggested to be involved in degradation of processed cTPs; Zn-oligopeptidase A, homologue of a peptidase that in E. coli was suggested to degrade small peptides down-stream of the Clp protease system; TPPII, tripeptyl peptidase.
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pone-0001994-g004: Quantification of the chloroplast protein homeostasis network including processing, (un)folding, maturation and proteolysis.Relative concentrations of 48 stromal proteins involved in the post-translational protein homeostasis network are displayed with color coding. Abbreviations are as follows: SPP, general stromal processing peptidase; PDF1A,B, methionine deformylases 1A,B; MSRA4,B2, methionine sulfoxide reductases; AP, amino-peptidases; CPN10,20,60, chaperone 10,20 and 60 of the GroEL/ES system; cpHSP70, heat shock protein 70; GrpE, nucleotide exchange factor; HSP90, heat shock protein 90; ClpB3, chaperone B3; cpSRP – chloroplast signal recognition particle subunit 43 and 54, involved in protein targeting components cpSRP43, cpSRP54; cpSecA – ATP-dependent Sec targeting component; cpTIG, a homologue of E. coli trigger factor involved in protein folding at the ribosome; ROC4, protein isomerase with unknown function; ClpP/R/S,T,C,D- subunits of the complete Clp protease system, DegP2 – protease of the Deg family; AtPrep1 - a Zn-protease suggested to be involved in degradation of processed cTPs; Zn-oligopeptidase A, homologue of a peptidase that in E. coli was suggested to degrade small peptides down-stream of the Clp protease system; TPPII, tripeptyl peptidase.

Mentions: To obtain better insight in the role of the quantified stromal proteome, all proteins were (re)evaluated for function, using information from papers, functional protein domain predictions, and other resources (e.g.TAIR). We used the MapMan bin system [45] for functional classification. As compared to previous chloroplast proteome studies, the current study significantly increased coverage of lower abundant pathways. Examples are nucleotide synthesis and degradation and nucleotide transfer, represented by 22 proteins out of the 39 cTP predicted plastid proteins assigned to this pathway, with an average abundance (log10(NAF)), of −3.4. Also, a set of 14 low abundant t-RNA synthetases were observed in the stroma with an average of −3.6, while soluble proteins involved in tetrapyrole biosynthesis have an average abundance of −3.3. The quantified stromal proteome also has a high number of proteins involved in protein translation, (un)folding, targeting, processing, aa modifications, and proteolysis. Here we highlight those stromal enzymes involved in the post-translational protein homeostasis network steps, including N-terminal processing and modifications (Figure 4).


Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.

Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson O, Sun Q, van Wijk KJ - PLoS ONE (2008)

Quantification of the chloroplast protein homeostasis network including processing, (un)folding, maturation and proteolysis.Relative concentrations of 48 stromal proteins involved in the post-translational protein homeostasis network are displayed with color coding. Abbreviations are as follows: SPP, general stromal processing peptidase; PDF1A,B, methionine deformylases 1A,B; MSRA4,B2, methionine sulfoxide reductases; AP, amino-peptidases; CPN10,20,60, chaperone 10,20 and 60 of the GroEL/ES system; cpHSP70, heat shock protein 70; GrpE, nucleotide exchange factor; HSP90, heat shock protein 90; ClpB3, chaperone B3; cpSRP – chloroplast signal recognition particle subunit 43 and 54, involved in protein targeting components cpSRP43, cpSRP54; cpSecA – ATP-dependent Sec targeting component; cpTIG, a homologue of E. coli trigger factor involved in protein folding at the ribosome; ROC4, protein isomerase with unknown function; ClpP/R/S,T,C,D- subunits of the complete Clp protease system, DegP2 – protease of the Deg family; AtPrep1 - a Zn-protease suggested to be involved in degradation of processed cTPs; Zn-oligopeptidase A, homologue of a peptidase that in E. coli was suggested to degrade small peptides down-stream of the Clp protease system; TPPII, tripeptyl peptidase.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2291561&req=5

pone-0001994-g004: Quantification of the chloroplast protein homeostasis network including processing, (un)folding, maturation and proteolysis.Relative concentrations of 48 stromal proteins involved in the post-translational protein homeostasis network are displayed with color coding. Abbreviations are as follows: SPP, general stromal processing peptidase; PDF1A,B, methionine deformylases 1A,B; MSRA4,B2, methionine sulfoxide reductases; AP, amino-peptidases; CPN10,20,60, chaperone 10,20 and 60 of the GroEL/ES system; cpHSP70, heat shock protein 70; GrpE, nucleotide exchange factor; HSP90, heat shock protein 90; ClpB3, chaperone B3; cpSRP – chloroplast signal recognition particle subunit 43 and 54, involved in protein targeting components cpSRP43, cpSRP54; cpSecA – ATP-dependent Sec targeting component; cpTIG, a homologue of E. coli trigger factor involved in protein folding at the ribosome; ROC4, protein isomerase with unknown function; ClpP/R/S,T,C,D- subunits of the complete Clp protease system, DegP2 – protease of the Deg family; AtPrep1 - a Zn-protease suggested to be involved in degradation of processed cTPs; Zn-oligopeptidase A, homologue of a peptidase that in E. coli was suggested to degrade small peptides down-stream of the Clp protease system; TPPII, tripeptyl peptidase.
Mentions: To obtain better insight in the role of the quantified stromal proteome, all proteins were (re)evaluated for function, using information from papers, functional protein domain predictions, and other resources (e.g.TAIR). We used the MapMan bin system [45] for functional classification. As compared to previous chloroplast proteome studies, the current study significantly increased coverage of lower abundant pathways. Examples are nucleotide synthesis and degradation and nucleotide transfer, represented by 22 proteins out of the 39 cTP predicted plastid proteins assigned to this pathway, with an average abundance (log10(NAF)), of −3.4. Also, a set of 14 low abundant t-RNA synthetases were observed in the stroma with an average of −3.6, while soluble proteins involved in tetrapyrole biosynthesis have an average abundance of −3.3. The quantified stromal proteome also has a high number of proteins involved in protein translation, (un)folding, targeting, processing, aa modifications, and proteolysis. Here we highlight those stromal enzymes involved in the post-translational protein homeostasis network steps, including N-terminal processing and modifications (Figure 4).

Bottom Line: The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude.Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction.No evidence was found for suggested targeting via the secretory system.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models.

Show MeSH
Related in: MedlinePlus