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.

Studying proteases and protein turnover in Arabidopsis chloroplasts.

Proteolysis is a key process for maintaining homeostasis in all living cells. The ability to degrade specific metabolic enzymes and regulatory proteins is essential for both cellular integrity and function. Equally important is the efficient removal of damaged or otherwise inactive polypeptides, especially during periods of developmental change or stress adaptation. Being one of the most metabolically active plant organelles, chloroplasts require various proteases to control overall protein quality. Much has been revealed about these chloroplast proteases over the last decade, and yet the identity of their native protein substrates remains elusive. In this chapter, we describe a variation upon a classic genetic approach to identify protease substrates based on the comparative protein degradation rates in wild-type and transgenic lines with impaired proteolytic activity. We have successfully used this approach with an in organello assay to identify numerous substrates for the stromal Clp protease from Arabidopsis thaliana, using both gene knockout mutants and antisense repression lines. In principle, the technique can be readily adapted for the study of other chloroplast proteases, and in other plant and algal species as the necessary genetic resources become available.

Assembly of the chloroplast ATP-dependent Clp protease in Arabidopsis is regulated by the ClpT accessory proteins.

The ATP-dependent caseinolytic protease (Clp) is an essential housekeeping enzyme in plant chloroplasts. It is by far the most complex of all known Clp proteases, with a proteolytic core consisting of multiple catalytic ClpP and noncatalytic ClpR subunits. It also includes a unique form of Clp protein of unknown function designated ClpT, two of which exist in the model species Arabidopsis thaliana. Inactivation of ClpT1 or ClpT2 significantly reduces the amount of Clp proteolytic core, whereas loss of both proves seedling lethal under autotrophic conditions. During assembly of the Clp proteolytic core, ClpT1 first binds to the P-ring (consisting of ClpP3-6 subunits) followed by ClpT2, and only then does the P-ring combine with the R-ring (ClpP1, ClpR1-4 subunits). Most of the ClpT proteins in chloroplasts exist in vivo as homodimers, which then apparently monomerize prior to association with the P-ring. Despite their relative abundance, however, the availability of both ClpT proteins is rate limiting for the core assembly, with the addition of recombinant ClpT1 and ClpT2 increasing core content up to fourfold. Overall, ClpT appears to regulate the assembly of the chloroplast Clp protease, revealing a new and sophisticated control mechanism on the activity of this vital protease in plants.

'happy on norflurazon' (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon-treated seedlings.

Various mutant screens have been undertaken to identify constituents involved in the transmission of signals from the plastid to the nucleus. Many of these screens have been performed using carotenoid-deficient plants grown in the presence of norflurazon (NF), an inhibitor of phytoene desaturase. NF-treated plants are bleached and suppress the expression of nuclear genes encoding chloroplast proteins. Several genomes uncoupled (gun) mutants have been isolated that de-repress the expression of these nuclear genes. In the present study, a genetic screen has been established that circumvents severe photo-oxidative stress in NF-treated plants. Under these modified screening conditions, happy on norflurazon (hon) mutants have been identified that, like gun mutants, de-repress expression of the Lhcb gene, encoding a light-harvesting chlorophyll protein, but, in contrast to wild-type and gun mutants, are green in the presence of NF. hon mutations disturb plastid protein homeostasis, thereby activating plastid signaling and inducing stress acclimatization. Rather than defining constituents of a retrograde signaling pathway specifically associated with the NF-induced suppression of nuclear gene expression, as proposed for gun, hon mutations affect Lhcb expression more indirectly prior to initiation of plastid signaling in NF-treated seedlings. They pre-condition seedlings by inducing stress acclimatization, thereby attenuating the impact of a subsequent NF treatment.

Identification of new protein substrates for the chloroplast ATP-dependent Clp protease supports its constitutive role in Arabidopsis.

The ATP-dependent Clp protease in plant chloroplasts consists of a heterogeneous proteolytic core containing multiple ClpP and ClpR paralogues. In this study, we have examined in detail the only viable knockout mutant to date of one of these subunits in Arabidopsis thaliana, ClpR1. Loss of ClpR1 caused a slow-growth phenotype, with chlorotic leaves during early development that later partially recovered upon maturity. Analysis of the Clp proteolytic core in the clpR1 mutant (clpR1-1) revealed approx. 10% of the wild-type levels remaining, probably due to a relative increase in the closely related ClpR3 protein and its partial substitution of ClpR1 in the core complex. A proteomic approach using an in organello proteolytic assay revealed 19 new potential substrates for the chloroplast Clp protease. Many of these substrates were constitutive enzymes involved in different metabolic pathways, including photosynthetic carbon fixation, nitrogen metabolism and chlorophyll/haem biosynthesis, whereas others function in housekeeping roles such as RNA maturation, protein synthesis and maturation, and recycling processes. In contrast, degradation of the stress-related chloroplast proteins Hsp21 (heat-shock protein 21) and lipoxygenase 2 was unaffected in the clpR1-1 line and thus not facilitated by the Clp protease. Overall, we show that the chloroplast Clp protease is principally a constitutive enzyme that degrades numerous stromal proteins, a feature that almost certainly underlies its vital importance for chloroplast function and plant viability.

An Arabidopsis thaliana virescent mutant reveals a role for ClpR1 in plastid development.

The ATP-dependent Clp protease has been well-characterized in Escherichia coli, but knowledge of its function in higher plants is limited. In bacteria, this two-component protease consists of a Ser-type endopeptidase ClpP, which relies on the ATP-dependent unfolding activity from an Hsp100 molecular chaperone to initiate protein degradation. In the chloroplasts of higher plants, multiple isoforms of the proteolytic subunit exist, with Arabidopsis having five ClpPs and four ClpP-like proteins termed ClpR predicted in its genome. In this work we characterized an Arabidopsis mutant impaired in one subunit of the chloroplast-localized Clp protease core, ClpR1. clpR1-1, a virescent mutant, carries a pre-mature stop codon in the clpR1 gene, resulting in no detectable ClpR1 protein. The accumulation of several chloroplast proteins, as well as most of the chloroplast-localized Clp protease subunits, is inhibited in clpR1-1. Unexpectedly, some plastid-encoded proteins do not accumulate, although their transcripts accumulate to wild-type levels. Maturation of 23S and 4.5S chloroplast ribosomal RNA (cp-rRNA) is delayed in clpR1-1, and both RNAs accumulate as higher molecular weight precursors. Also, chloroplasts in clpR1-1 are smaller than in wild type and have fewer thylakoid membranes with smaller grana stacks. We propose that a ClpR1-containing activity is required for chloroplast development and differentiation and in its absence both are delayed.

Structural and functional insights into the chloroplast ATP-dependent Clp protease in Arabidopsis.

In contrast with the model Escherichia coli Clp protease, the ATP-dependent Clp protease in higher plants has a remarkably diverse proteolytic core consisting of multiple ClpP and ClpR paralogs, presumably arranged within a dual heptameric ring structure. Using antisense lines for the nucleus-encoded ClpP subunit, ClpP6, we show that the Arabidopsis thaliana Clp protease is vital for chloroplast development and function. Repression of ClpP6 produced a proportional decrease in the Clp proteolytic core, causing a chlorotic phenotype in young leaves that lessened upon maturity. Structural analysis of the proteolytic core revealed two distinct subcomplexes that likely correspond to single heptameric rings, one containing the ClpP1 and ClpR1-4 proteins, the other containing ClpP3-6. Proteomic analysis revealed several stromal proteins more abundant in clpP6 antisense lines, suggesting that some are substrates for the Clp protease. A proteolytic assay developed for intact chloroplasts identified potential substrates for the stromal Clp protease in higher plants, most of which were more abundant in young Arabidopsis leaves, consistent with the severity of the chlorotic phenotype observed in the clpP6 antisense lines. The identified substrates all function in more general housekeeping roles such as plastid protein synthesis, folding, and quality control, rather than in metabolic activities such as photosynthesis.

Inactivation of the clpC1 gene encoding a chloroplast Hsp100 molecular chaperone causes growth retardation, leaf chlorosis, lower photosynthetic activity, and a specific reduction in photosystem content.

ClpC is a molecular chaperone of the Hsp100 family. In higher plants there are two chloroplast-localized paralogs (ClpC1 and ClpC2) that are approximately 93% similar in primary sequence. In this study, we have characterized two independent Arabidopsis (Arabidopsis thaliana) clpC1 T-DNA insertion mutants lacking on average 65% of total ClpC content. Both mutants display a retarded-growth phenotype, leaves with a homogenous chlorotic appearance throughout all developmental stages, and more perpendicular secondary influorescences. Photosynthetic performance was also impaired in both knockout lines, with relatively fewer photosystem I and photosystem II complexes, but no changes in ATPase and Rubisco content. However, despite the specific drop in photosystem I and photosystem II content, no changes in leaf cell anatomy or chloroplast ultrastructure were observed in the mutants compared to the wild type. Previously proposed functions for envelope-associated ClpC in chloroplast protein import and degradation of mistargeted precursors were examined and shown not to be significantly impaired in the clpC1 mutants. In the stroma, where the majority of ClpC protein is localized, marked increases of all ClpP paralogs were observed in the clpC1 mutants but less variation for the ClpR paralogs and a corresponding decrease in the other chloroplast-localized Hsp100 protein, ClpD. Increased amounts of other stromal molecular chaperones (Cpn60, Hsp70, and Hsp90) and several RNA-binding proteins were also observed. Our data suggest that overall ClpC as a stromal molecular chaperone plays a vital role in chloroplast function and leaf development and is likely involved in photosystem biogenesis.