Quantitative Analysis of the Chloroplast Molecular Chaperone ClpC/Hsp93 in Arabidopsis Reveals New Insights into Its Localization,Interaction with the Clp Proteolytic Core,and Functional Importance

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.     

Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane.     

Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC

The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones-regulators of proteolysis. By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC. The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis. Northern blot experiments showed that expression of clpC was induced in response to high temperature (40-42 degrees C) versus 37 degrees C, suggesting that ClpC is a heat shock protein. Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases. The clpC-deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin. The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells. Finally, the clpC disruption resulted in decreased genetic transformation. Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline-binding proteins LytA, CbpA, CbpE, CbpF, CbpJ. These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.     

Downregulation of ClpR2 leads to reduced accumulation of the ClpPRS protease complex and defects in chloroplast biogenesis in Arabidopsis

Plastids contain tetradecameric Clp protease core complexes, with five ClpP Ser-type proteases, four nonproteolytic ClpR, and two associated ClpS proteins. Accumulation of total ClpPRS complex decreased twofold to threefold in an Arabidopsis thaliana T-DNA insertion mutant in CLPR2 designated clpr2-1. Differential stable isotope labeling of the ClpPRS complex with iTRAQ revealed a fivefold reduction in assembled ClpR2 accumulation and twofold to fivefold reductions in the other subunits. A ClpR2:(his)(6) fusion protein that incorporated into the chloroplast ClpPRS complex fully complemented clpr2-1. The reduced accumulation of the ClpPRS protease complex led to a pale-green phenotype with delayed shoot development, smaller chloroplasts, decreased thylakoid accumulation, and increased plastoglobule accumulation. Stromal ClpC1 and 2 were both recruited to the thylakoid surface in clpr2-1. The thylakoid membrane of clpr2-1 showed increased carotenoid content, partial inactivation of photosystem II, and upregulated thylakoid proteases and stromal chaperones, suggesting an imbalance in chloroplast protein homeostasis and a well-coordinated network of proteolysis and chaperone activities. Interestingly, a subpopulation of PsaF and several light-harvesting complex II proteins accumulated in the thylakoid with unprocessed chloroplast transit peptides. We conclude that ClpR2 cannot be functionally replaced by other ClpP/R homologues and that the ClpPRS complex is central to chloroplast biogenesis, thylakoid protein homeostasis, and plant development.     

Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis

cpSRP54 (for chloroplast SIGNAL RECOGNITION PARTICLE54) is involved in cotranslational and posttranslational sorting of thylakoid proteins. The Arabidopsis (Arabidopsis thaliana) cpSRP54 null mutant, ffc1-2, is pale green with delayed development. Western-blot analysis of individual leaves showed that the SRP sorting pathway, but not the SecY/E translocon, was strongly down-regulated with progressive leaf development in both wild-type and ffc1-2 plants. To further understand the impact of cpSRP54 deletion, a quantitative comparison of ffc2-1 was carried out for total leaf proteomes of young seedlings and for chloroplast proteomes of fully developed leaves using stable isotope labeling (isobaric stable isotope labeling and isotope-coded affinity tags) and two-dimensional gels. This showed that cpSRP54 deletion led to a change in light-harvesting complex composition, an increase of PsbS, and a decreased photosystem I/II ratio. Moreover, the cpSRP54 deletion led in young leaves to up-regulation of thylakoid proteases and stromal chaperones, including ClpC. In contrast, the stromal protein homeostasis machinery returned to wild-type levels in mature leaves, consistent with the developmental down-regulation of the SRP pathway. A differential response between young and mature leaves was also found in carbon metabolism, with an up-regulation of the Calvin cycle and the photorespiratory pathway in peroxisomes and mitochondria in young leaves but not in old leaves. The Calvin cycle was down-regulated in mature leaves to adjust to the reduced capacity of the light reaction, while reactive oxygen species defense proteins were up-regulated. The significance of ClpC up-regulation was confirmed through the generation of an ffc2-1 clpc1 double mutant. This mutant was seedling lethal under autotrophic conditions but could be partially rescued under heterotrophic conditions.     

The amino-terminal domain of chloroplast Hsp93 is important for its membrane association and functions in vivo

Chloroplast 93-kD heat shock protein (Hsp93/ClpC), an Hsp100 family member, is suggested to have various functions in chloroplasts, including serving as the regulatory chaperone for the ClpP protease in the stroma and acting as a motor component of the protein translocon at the envelope. Indeed, although Hsp93 is a soluble stromal protein, a portion of it is associated with the inner envelope membrane. The mechanism and functional significance of this Hsp93 membrane association have not been determined. Here, we mapped the region important for Hsp93 membrane association by creating various deletion constructs and found that only the construct with the amino-terminal domain deleted, Hsp93-ΔN, had reduced membrane association. When transformed into Arabidopsis (Arabidopsis thaliana), most atHsp93V-ΔN proteins did not associate with membranes and atHsp93V-ΔΝ failed to complement the pale-green and protein import-defective phenotypes of an hsp93V knockout mutant. The residual atHsp93V-ΔN at the membranes had further reduced association with the central protein translocon component Tic110. However, the degradation of chloroplast glutamine synthetase, a potential substrate for the ClpP protease, was not affected in the hsp93V mutant or in the atHSP93V-ΔN transgenic plants. Hsp93-ΔN also had the same ATPase activity as that of full-length Hsp93. These data suggest that the association of Hsp93 with the inner envelope membrane through its amino-terminal domain is important for the functions of Hsp93 in vivo.     

The thylakoid protease Deg2 is involved in stress-related degradation of the photosystem II light-harvesting protein Lhcb6 in Arabidopsis thaliana

? The thylakoid protease Deg2 is a serine-type protease peripherally attached to the stromal side of the thylakoid membrane. Given the lack of knowledge concerning its function, two T-DNA insertion lines devoid of Deg2 were prepared to study the functional importance of this protease in Arabidopsis thaliana. ? The phenotypic appearance of deg2 mutants was studied using a combination of stereo and transmission electron microscopy, and short-stress-mediated degradation of apoproteins of minor light-harvesting antennae of photosystem II (PSII) was analysed by immunoblotting in the mutants in comparison with wild-type plants. ? Deg2 repression produced a phenotype in which reduced leaf area and modified chloroplast ultrastructure of older leaves were the most prominent features. In contrast to the wild type, the chloroplasts of second-whorl leaves of 4-wk-old deg2 mutants did not display features typical of the early senescence phase, such as undulation of the chloroplast envelope and thylakoids. The ability to degrade the photosystem II light-harvesting protein Lhcb6 apoprotein in response to brief high-salt, wounding, high-temperature and high-irradiance stress was demonstrated to be impaired in deg2 mutants. ? Our results suggest that Deg2 is required for normal plant development, including the chloroplast life cycle, and has an important function in the degradation of Lhcb6 in response to short-duration stresses.     

The Arabidopsis chloroplastic NifU-like protein CnfU, which can act as an iron-sulfur cluster scaffold protein, is required for biogenesis of ferredoxin and photosystem I

The biosynthesis of iron-sulfur clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of iron-sulfur clusters. Here, we report the identification of two chloroplast-localized NifU-like proteins, AtCnfU-V and AtCnfU-IVb, from Arabidopsis (Arabidopsis thaliana) with high sequence similarity to a cyanobacterial NifU-like protein that was proposed to serve as a molecular scaffold. AtCnfU-V is constitutively expressed in several tissues of Arabidopsis, whereas the expression of AtCnfU-IVb is prominent in the aerial parts. Mutant Arabidopsis lacking AtCnfU-V exhibited a dwarf phenotype with faint pale-green leaves and had drastically impaired photosystem I accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of ferredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vitro activity of iron-sulfur cluster insertion into apo-ferredoxin. When expressed in Escherichia coli cells, AtCnfU-V formed a homodimer carrying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-ferredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis in chloroplasts and thereby is required for biogenesis of ferredoxin and photosystem I.     

千里光热激蛋白90-3(Hsp90-3)的生物信息学与功能分析

Hsp90是真核细胞重要的一类分子伴侣,与植物的生长发育、抗逆性、信号转导及生物进化等功能密切相关.为了深入理解高等植物Hsp90结构与功能的关系,该研究从千里光(Senecio scandens)全长cDNA文库中分离到Hsp90-3基因.序列分析结果表明,该基因编码699个氨基酸的多肽,与拟南芥(Arabidopsis thaliana)AtHsp90-3(登录号:NP_200412.1)的同源性最高,为93.71％;预测蛋白质的分子量为79.78 kD,理论等电点为5.08.信号序列分析结果发现,该蛋白主要定位于细胞的细胞核、过氧化物酶体、叶绿体类囊体膜及叶绿体基质中,提示作为分子伴侣,高等植物Hsp90-3参与细胞内膜系统蛋白质的转运.结构与功能分析发现,该蛋白有3个结构域及1个连接区,推测Hsp90-3在真核细胞的信号转导、转录调控及胁迫表达等过程中发挥重要功能.     

Disruption and analysis of the clpB, clpC, and clpE genes in Lactococcus lactis: ClpE, a new Clp family in gram-positive bacteria

In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes (clpC, clpE, and clpB) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX2CX22CX2C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by clpB was strongly induced by heat shock and, while clpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in clpB, clpC, or clpE, we found that although the genes are part of the L. lactis heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins, clpE mutant cells formed smaller colonies than wild-type cells and clpB and clpC mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in L. lactis, could be necessary for degrading proteins generated by certain types of stress.     

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts

Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system.     

The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins

The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress in Bacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased in clpC, clpP, and clpX mutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections in clpC and clpP mutant cells even under nonstress conditions. In contrast, in the wild type or clpX mutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption that clpC and clpP mutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.     

In vivo studies on the roles of Tic110, Tic40 and Hsp93 during chloroplast protein import

A multisubunit translocon of the inner envelope membrane, termed Tic, mediates the late stages of protein import into chloroplasts. Membrane proteins, Tic110 and Tic40, and a stromal chaperone, Hsp93, have been proposed to function together within the Tic complex. In Arabidopsis, single genes, atTIC110 and atTIC40, encode the Tic proteins, and two homologous genes, atHSP93-V and atHSP93-III, encode Hsp93. These four genes exhibited relatively uniform patterns of expression, suggesting important roles for plastid biogenesis throughout development and in all tissues. To investigate the roles played by these proteins in vivo, we conducted a comparative study of T-DNA knockout mutants for each Tic gene, and for the most abundantly expressed Hsp93 gene, atHSP93-V. In the homozygous state, the tic110 mutation caused embryo lethality, implying an essential role for atTic110 during plastid biogenesis. Homozygous tic110 embryos exhibited retarded growth, developmental arrest at the globular stage and a 'raspberry-like' embryo-proper phenotype. Heterozygous tic110 plants, and plants homozygous for the tic40 and hsp93-V mutations, exhibited chlorosis, aberrant chloroplast biogenesis, and inefficient chloroplast-import of both photosynthetic and non-photosynthetic preproteins. Non-additive interactions amongst the mutations occurred in double mutants, suggesting that the three components may cooperate during chloroplast protein import.     

Protective function of chloroplast 2-cysteine peroxiredoxin in photosynthesis. Evidence from transgenic Arabidopsis

2-Cysteine peroxiredoxins (2-CPs) constitute a ubiquitous group of peroxidases that reduce cell-toxic alkyl hydroperoxides to their corresponding alcohols. Recently, we cloned 2-CP cDNAs from plants and characterized them as chloroplast proteins. To elucidate the physiological function of the 2-CP in plant metabolism, we generated antisense mutants in Arabidopsis. In the mutant lines a 2-CP deficiency developed during early leaf and plant development and eventually the protein accumulated to wild-type levels. In young mutants with reduced amounts of 2-CP, photosynthesis was impaired and the levels of D1 protein, the light-harvesting protein complex associated with photosystem II, chloroplast ATP synthase, and ribulose-1,5-bisphosphate carboxylase/oxygenase were decreased. Photoinhibition was particularly pronounced after the application of the protein synthesis inhibitor, lincomycin. We concluded that the photosynthetic machinery needs high levels of 2-CP during leaf development to protect it from oxidative damage and that the damage is reduced by the accumulation of 2-CP protein, by the de novo synthesis and replacement of damaged proteins, and by the induction of other antioxidant defenses in 2-CP mutants.     

Interaction of plant mitochondrial and chloroplast signal peptides with the Hsp70 molecular chaperone.

Most mitochondrial and chloroplast proteins are synthesized on cytosolic polyribosomes as precursor proteins, with an N-terminal signal sequence that targets the precursor to the correct organelle. In mitochondria, the chaperone Hsp70 functions as a molecular motor, pulling the precursor across the mitochondrial membranes; 97.0% of plant mitochondrial presequences contain an Hsp70 binding site. In chloroplasts, the outer envelope, intermembrane space and a stromal Hsp70 are thought to participate in protein import; 82.5% of chloroplast transit peptides have an Hsp70 binding site. The interaction of signal peptides with Hsp70 during the import process is supported by biochemical and bioinformatic studies.