Identification of a 350-kDa ClpP Protease Complex with 10 Different Clp Isoforms in Chloroplasts of Arabidopsis thaliana

Peltier, Jean‐Benoǐt; Ytterberg, A. Jimmy; Liberles, David A.; Roepstorff, Peter; Wijk, Klaas J. van

doi:10.1074/jbc.m010503200

Cited by 108 publications

(115 citation statements)

References 46 publications

(42 reference statements)

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“…ClpP2 was found in mitochondria by proteomic analyses as well (Millar et al, 2001;Peltier et al, 2004). Other ClpPs and ClpRs were found only in chloroplasts (Sokolenko et al, 1998;Nakabayashi et al, 1999;Peltier et al, 2001Peltier et al, , 2004Zheng et al, 2002). The pea homolog of FtsH3 was detected in mitochondria but not in chloroplasts (Kolodziejczak et al, 2002).…”

Section: Discussionmentioning

confidence: 85%

“…With the exception of ClpP1, which is encoded in the plastid genome, all other genes are nuclear. Most of ClpP isomers, as well as their proteolytically inactive homologs ClpR, are found in chloroplasts, where two isomers of ClpC and one ClpD, the plant homologs of ClpA, are also located (Peltier et al, 2001;Zheng et al, 2002). One isozyme, ClpP2, is found in mitochondria, where it may associate with the regulatory subunit ClpX (Halperin et al, 2001b;Peltier et al, 2004).…”

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confidence: 99%

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Expression in Multigene Families. Analysis of Chloroplast and Mitochondrial Proteases

Sinvany-Villalobo

Davydov²,

Ben-Ari³

et al. 2004

Plant Physiology

119

107

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The proteolytic machinery of chloroplasts and mitochondria in Arabidopsis consists primarily of three families of ATPdependent proteases, Clp, Lon, and FtsH, and one family of ATP-independent proteases, DegP. However, the functional significance of the multiplicity of their genes is not clear. To test whether expression of specific isomers could be differently affected by growth conditions, we analyzed transcript abundance following short-term exposure to different environmental stimuli, using 70-mer oligonucleotide arrays. This analysis revealed variability in the response to high light and different temperatures within members of each family. Thirty out of the 41 tested genes were up-regulated in response to high light, including both chloroplast and mitochondrial isozymes, whereas only six and five genes responded to either high or low temperature, respectively. The extent of response was variable, ranging from 2-to 20-fold increase in the steady-state levels.Absolute transcript levels of the tested genes, compiled from one-channel arrays, were also variable. In general, transcripts encoding mitochondrial isozymes were accumulated to a lower level than chloroplastic ones. Within the FtsH family, transcript abundance of most genes correlated with the severity of mutant phenotypes in the relevant genes. This correlation was also evident at the protein level. Analysis of FtsH isozymes revealed that FtsH2 was the most abundant species, followed by FtsH5 and 8, with FtsH1 being accumulated to only 10% of FtsH2 level. These results suggest that, unlike previous expectations, the relative importance of different chloroplast protease isozymes, evidenced by mutant phenotypes at least in the FtsH family, is determined by their abundance, and not necessarily by different specific functions or specialized expression under certain conditions.The proteolytic machinery of chloroplasts and mitochondria is essential for controlling the quality and turnover of these organelles' proteins and, thus, is important for their proper function. In Arabidopsis, the proteolytic machinery of chloroplasts consists primarily of three families of ATP-dependent proteases, Clp, Lon, and FtsH, and one family of ATPindependent proteases, DegP (for review, see Adam and Clarke, 2002;Sokolenko et al., 2002). Homologous enzymes are found also in mitochondria (Sarria et al., 1998;Adam et al., 2001;Halperin et al., 2001b;Kolodziejczak et al., 2002). All these families have well-characterized homologs in Escherichia coli (for review, see Clarke, 1999;Adam, 2000). Clp is a Ser protease that separates its two essential functions in two different polypeptides: a small subunit, ClpP, containing the proteolytic active site, and a larger regulatory ATPase subunit, either ClpA or ClpX (for review, see Gottesman, 1996). Lon protease is an ATPdependent Ser protease in which the catalytic and ATPase domains reside in a single polypeptide (for review, see Gottesman, 1996). FtsH is the only essential ATP-dependent protease in E. coli. It is a membranebound metall...

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Section: Discussionmentioning

confidence: 85%

mentioning

confidence: 99%

Expression in Multigene Families. Analysis of Chloroplast and Mitochondrial Proteases

Sinvany-Villalobo

Davydov²,

Ben-Ari³

et al. 2004

Plant Physiology

119

107

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“…Native gel separation of the chloroplast stroma proteome indicated that the Clp core complex migrates at 350 kD (Peltier et al, 2001(Peltier et al, , 2004 and its component rings at 180 to 230 kD (Sjö gren et al, 2006). Native isoelectric focusing further showed that the plastid ClpPR subunits form a single core complex (Peltier et al, 2004).…”

Section: Affinity Purification and Ms-based Characterization Of Chlormentioning

confidence: 99%

“…Previous phylogenetic analyses of the Clp family (Peltier et al, 2001;Majeran et al, 2005) highlighted the evolutionary linkages among the Clp subunits in cyanobacterial and algal species with Arabidopsis and tobacco (Nicotiana tabacum). To better understand the evolution and adaptation of the Clp core complex in plants, we took advantage of the recently sequenced plant genomes from the monocotelydons maize (Zea mays) and rice (Oryza sativa) and dicotyledons Arabidopsis and poplar (both Rosales), in addition to the moss Physcomitrella patens (Bryophyta) and the spikemoss Selaginella moellendorffii (Lycopodiophyta).…”

Section: Evolutionary Relationships Among Clp Subunitsmentioning

confidence: 99%

Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis

et al. 2011

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The caseinolytic protease (Clp) protease system has been expanded in plant plastids compared with its prokaryotic progenitors. The plastid Clp core protease consists of five different proteolytic ClpP proteins and four different noncatalytic ClpR proteins, with each present in one or more copies and organized in two heptameric rings. We determined the exact subunit composition and stoichiometry for the intact core and each ring. The chloroplast ClpP/R protease was affinity purified from clpr4 and clpp3 Arabidopsis thaliana null mutants complemented with C-terminal StrepII-tagged versions of CLPR4 and CLPP3, respectively. The subunit stoichiometry was determined by mass spectrometry-based absolute quantification using stable isotope-labeled proteotypic peptides generated from a synthetic gene. One heptameric ring contained ClpP3,4,5,6 in a 1:2:3:1 ratio. The other ring contained ClpP1 and ClpR1,2,3,4 in a 3:1:1:1:1 ratio, resulting in only three catalytic sites. These ClpP1/R1-4 proteins are most closely related to the two subunits of the cyanobacterial P3/R complex and the identical P:R ratio suggests conserved adaptation. Furthermore, the plant-specific C-terminal extensions of the ClpP/R subunits were not proteolytically removed upon assembly, suggesting a regulatory role in Clp chaperone interaction. These results will now allow testing ClpP/R structure-function relationships using rationale design. The quantification workflow we have designed is applicable to other protein complexes.

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“…Six genes encoding ClpP, 2 encoding ClpC, 11 encoding FtsH, and 13 encoding DegP have been identified Peltier et al 2001). The products of most of these genes are expected to be targeted to chloroplasts.…”

Section: Introductionmentioning

confidence: 99%

A single recessive mutation in the proteolytic machinery of Arabidopsis chloroplasts impairs photoprotection and photosynthesis upon cold stress

Levy

Bachmair

Adam

2004

Planta

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Intracellular proteases, together with molecular chaperones, are components of the cellular protein quality control system. Although the identity of chloroplast proteases has been revealed in recent years, little is known about their regulation. As a first step towards identifying unknown functional or regulatory components of the chloroplast proteolytic machinery, a genetic screen was devised with the aim of generating Arabidopsis thaliana (L.) Heynh. mutants impaired in chloroplast protein degradation. A streptomycin-resistance gene was fused to a cDNA construct that encodes an unstable mutant of the OE33 protein. This chimeric gene was used for transforming Arabidopsis plants. Analysis of transgenic plants revealed a correlation between streptomycin resistance and accumulation of the recombinant fusion protein. Seeds from transgenic plants that were sensitive to streptomycin were chemically mutagenized and screened for resistance to streptomycin. Such resistance could be due to stabilization of the protein caused by a mutation in the chloroplast proteolytic machinery. Genetic analysis of one of the mutants showed the mutation to be recessive, in a single nuclear gene. Further characterization of the mutant revealed that it was not a result of increased transcription of the transgene. Moreover, chloroplast lysates from the mutant plant showed decreased ATP-dependent degradation of a chloroplast protein substrate, consistent with the conclusion that the mutation impaired the proteolytic machinery. Physiological analysis revealed that upon cold stress, photoprotection and photosynthesis in the mutant was inhibited, suggesting that the chloroplast proteolytic machinery is involved in repairing damage incurred to the photosynthetic machinery upon exposure to cold temperatures.

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Identification of a 350-kDa ClpP Protease Complex with 10 Different Clp Isoforms in Chloroplasts of Arabidopsis thaliana

Cited by 108 publications

References 46 publications

Expression in Multigene Families. Analysis of Chloroplast and Mitochondrial Proteases

Expression in Multigene Families. Analysis of Chloroplast and Mitochondrial Proteases

Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis

A single recessive mutation in the proteolytic machinery of Arabidopsis chloroplasts impairs photoprotection and photosynthesis upon cold stress

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