Structures, Functions, and Interactions of ClpT1 and ClpT2 in the Clp Protease System of Arabidopsis Chloroplasts

Kim, Ji-Tae; Kimber, Matthew S.; Nishimura, Kenji; Friso, Giulia; Schultz, Lance; Ponnala, Lalit; Wijk, Klaas J. van

doi:10.1105/tpc.15.00106

Cited by 44 publications

(88 citation statements)

References 57 publications

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“…ClpP2 could have a yet-to-be-characterized prosequence or require another type of modification, such as phosphorylation or alkylation, to assemble and associate with ClpA or ClpX. Or, ClpP2 could require a protein or small-molecule cofactor for assembly, just as ClpCP from Bacillus subtilis needs MecA for efficient assembly and ClpP/R from Arabidopsis thaliana chloroplasts needs ClpT1 and ClpT2 (38,39). Like a ClpP1/P2 heteromer, modified active ClpP2 might increase or decrease the efficiency of proteolysis or change the abundance of available proteases.…”

Section: Discussionmentioning

confidence: 99%

Two Isoforms of Clp Peptidase in Pseudomonas aeruginosa Control Distinct Aspects of Cellular Physiology

et al. 2017

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Caseinolytic peptidases (ClpPs) regulate diverse aspects of cellular physiology in bacteria. Some species have multiple ClpPs, including the opportunistic pathogen Pseudomonas aeruginosa, in which there is an archetypical isoform, ClpP1, and a second isoform, ClpP2, about which little is known. Here, we use phenotypic assays to investigate the biological roles of ClpP1 and ClpP2 and biochemical assays to characterize purified ClpP1, ClpP2, ClpX, and ClpA. Interestingly, ClpP1 and ClpP2 have distinct intracellular roles for motility, pigment production, iron scavenging, and biofilm formation. Of particular interest, ClpP2, but not ClpP1, is required for microcolony organization, where multicellular organized structures first form on the pathway to biofilm production. We found that purified ClpP1 with ClpX or ClpA was enzymatically active, yet to our surprise, ClpP2 was inactive and not fully assembled in vitro; attempts to assist ClpP2 assembly and activation by mixing with the other Clp components failed to turn on ClpP2, as did solution conditions that have helped activate other ClpPs in vitro. We postulate that the active form of ClpP2 has yet to be discovered, and we present several potential models to explain its activation as well as the unique role ClpP2 plays in the development of the clinically important biofilms in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is responsible for severe infections of immunocompromised patients. Our work demonstrates that two different isoforms of the Clp peptidase, ClpP1 and ClpP2, control distinct aspects of cellular physiology for this organism. In particular, we identify ClpP2 as being necessary for microcolony organization. Pure active forms of ClpP1 and either ClpX or ClpA were characterized as assembled and active, and ClpP2 was incompletely assembled and inactive. By establishing both the unique biological roles of ClpP1 and ClpP2 and their initial biochemical assemblies, we have set the stage for important future work on the structure, function, and biological targets of Clp proteolytic enzymes in this important opportunistic pathogen.

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Section: Discussionmentioning

confidence: 99%

Two Isoforms of Clp Peptidase in Pseudomonas aeruginosa Control Distinct Aspects of Cellular Physiology

et al. 2017

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“…Clp core assembly and stabilization require plant-specific accessory proteins ClpT1/2 (Peltier et al, 2004;Sjögren and Clarke, 2011;Clarke, 2012;Kim et al, 2015). Lossof-function mutants for the ClpC1 chaperone and the ClpPRT core show pale-green, seedling-lethal, or embryodefective phenotypes, whereas knockouts for two adaptor proteins and ClpC2/D display no visible effects, underscoring their distinct contributions to plant development.…”

Section: Clpmentioning

confidence: 99%

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments

2016

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ORCID IDs: 0000-0003-1718-9121 (Y.K.); 0000-0001-9747-5042 (W.S.).Chloroplasts originated from the endosymbiosis of ancestral cyanobacteria and maintain transcription and translation machineries for around 100 proteins. Most endosymbiont genes, however, have been transferred to the host nucleus, and the majority of the chloroplast proteome is composed of nucleus-encoded proteins that are biosynthesized in the cytosol and then imported into chloroplasts. How chloroplasts and the nucleus communicate to control the plastid proteome remains an important question. Protein-degrading machineries play key roles in chloroplast proteome biogenesis, remodeling, and maintenance. Research in the past few decades has revealed more than 20 chloroplast proteases, which are localized to specific suborganellar locations. In particular, two energy-dependent processive proteases of bacterial origin, Clp and FtsH, are central to protein homeostasis. Processing endopeptidases such as stromal processing peptidase and thylakoidal processing peptidase are involved in the maturation of precursor proteins imported into chloroplasts by cleaving off the amino-terminal transit peptides. Presequence peptidases and organellar oligopeptidase subsequently degrade the cleaved targeting peptides. Recent findings have indicated that not only intraplastidic but also extraplastidic processive protein-degrading systems participate in the regulation and quality control of protein translocation across the envelopes. In this review, we summarize current knowledge of the major chloroplast proteases in terms of type, suborganellar localization, and diversification. We present details of these degradation processes as case studies according to suborganellar compartment (envelope, stroma, and thylakoids). Key questions and future directions in this field are discussed.Over 1 billion years of plastid evolution since the endosymbiosis of ancestral cyanobacteria (Douzery et al., 2004), chloroplast biogenesis has gained complexity, with large sets of the endosymbiont genes being transferred to host nuclear genomes. While only 100 endosymbiont genes remain in the plastid genome, with the corresponding proteins biosynthesized there, the nuclear genes have often gained complexity by duplication and diversification of the original endosymbiotic genes. This complexity raises numerous questions regarding (1) at what level gene expression is coordinately controlled, (2) what molecules coordinate the cross talk between chloroplasts and the nucleus, (3) how proteins get across membranes and become imported into chloroplasts, and (4) how the stoichiometries of nucleus-and chloroplast-encoded subunits within individual chloroplast protein complexes such as photosystems and Rubisco are strictly maintained.Given these questions, chloroplast biogenesis has remained a central subject in plant physiology for the last few decades (Jarvis and López-Juez, 2013). In our view, the aforementioned questions point to the importance of protein homeostasis and posttranslational modificat...

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“…noctiflora (88,166 sequences; 20,816,406 residues) and S . latifolia (101,108 sequences; 20,447,864 residues) was done using Mascot, followed by filtering, grouping of closely related sequences based on matched MS/MS spectra and quantification based on normalized AdjSPC (NadjSPC) as in (Friso et al ., 2011; Kim et al ., 2015). For each species, databases were a merger of annotated proteins from organellar genomes (Sloan, Alverson, Chuckalovcak, et al ., 2012; Sloan, Alverson, Wu, et al ., 2012) and protein sequence predictions generated by TransDecoder (Haas et al ., 2013) from transcriptome assemblies (Sloan, Triant, Wu, et al ., 2014).…”

Section: Methodsmentioning

confidence: 99%

“…For replicate 1, we used one gel lane for each species, whereas we pooled two gel lanes for replicate 2 to increase protein identifications and sequence coverage. The resuspended peptide extracts were analyzed by data-dependent MS/MS using an on-line LC-LTQ-Orbitrap (Thermo Electron Corp.) with details as described in (Kim et al ., 2015). Hence, a total of 38 MS/MS runs for each species was carried out.…”

Section: Methodsmentioning

confidence: 99%

Extreme variation in rates of evolution in the plastid Clp protease complex

Williams

Friso

Wijk

et al. 2018

Preprint

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1Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the 2 level of multisubunit complexes in mitochondria and plastids. One such complex in plants is the 3 caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The 4 proteolytic core of Clp comprises subunits from one plastid-encoded gene (clpP1) and multiple 5 nuclear genes. The clpP1 gene is highly conserved across most green plants, but it is by far the 6 fastest evolving plastid-encoded gene in some angiosperms. To better understand these extreme 7 and mysterious patterns of divergence, we investigated the history of clpP1 molecular evolution 8 across green plants by extracting sequences from 988 published plastid genomes. We find that 9 clpP1 has undergone remarkably frequent bouts of accelerated sequence evolution and 10 architectural changes (e.g., loss of introns and RNA-editing sites) within seed plants. Although 11 clpP1 is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that 12 this is rarely the case. We applied comparative native gel electrophoresis of chloroplast protein 13 complexes followed by protein mass spectrometry in two species within the angiosperm genus 14 Silene, which has highly elevated and heterogeneous rates of clpP1 evolution. We confirmed that 15 clpP1 is expressed as a stable protein and forms oligomeric complexes with the nuclear-encoded 16 Clp subunits, even in one of the most divergent Silene species. Additionally, there is a tight 17 correlation between amino-acid substitution rates in clpP1 and the nuclear-encoded Clp subunits 18 across a broad sampling of angiosperms, suggesting ongoing selection on interactions within this 19 complex. 21Rates of sequence evolution vary dramatically across genes and genomes. Understanding 22 the forces that determine such variation is one of the defining goals in the field of molecular 23 evolution. In seed plants, the plastid genome (plastome) generally evolves two-to six-fold more 24 slowly than the nuclear genome (Wolfe et al., 1987; Drouin et al., 2008; Smith and Keeling, 25 2015). However, among angiosperms, there is considerable heterogeneity in the rate of plastome 26 evolution. Many lineages have maintained a slowly-evolving plastome, while others have 27 experienced drastic rate increases (Jansen et al., 2007). For instance, among close relatives 28 within the tribe Sileneae there have been at least three recent and independent increases in 29 plastome evolutionary rate (Erixon and Oxelman, 2008; Sloan, Triant, Forrester, et al., 2014). 30 Similar accelerations have been documented in the Campanulaceae (Haberle et al., 2008; a structural level, plastome gene order has largely been conserved, with most angiosperms 34 retaining the structural organization that was present in the most recent common ancestor of this 35 group (Raubeson and Jansen, 2005). However, the sporadic increases in rates of plastome 36 sequence evolution have often been accompanied by structural ...

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Structures, Functions, and Interactions of ClpT1 and ClpT2 in the Clp Protease System of Arabidopsis Chloroplasts

Cited by 44 publications

References 57 publications

Two Isoforms of Clp Peptidase in Pseudomonas aeruginosa Control Distinct Aspects of Cellular Physiology

Two Isoforms of Clp Peptidase in Pseudomonas aeruginosa Control Distinct Aspects of Cellular Physiology

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments

Extreme variation in rates of evolution in the plastid Clp protease complex

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