Mutations in ClpC2/Hsp100 suppress the requirement for FtsH in thylakoid membrane biogenesis

Abstract

The Arabidopsis var2 variegation mutant defines a nuclear gene for a chloroplast FtsH metalloprotease. Leaf variegation is expressed only in homozygous recessive plants. The cells in the green leaf sectors of this mutant contain morphologically normal chloroplasts, whereas cells in the white sectors contain abnormal plastids lacking organized lamellar structures. var2 mutants are hypersusceptible to photoinhibition, and VAR2 degrades unassembled polypeptides and is involved in the D1 repair cycle of photosystem II, likely by affecting turnover of the photodamaged D1 polypeptide. A second-site suppressor screen of var2 yielded a normalappearing, nonvariegated line. Map-based cloning revealed that the suppression of variegation in this line is due to a splice site mutation in ClpC2, a chloroplast Hsp100 chaperone, that results in sharply reduced ClpC2 protein accumulation. Isolation of clpC2 single mutants showed that clpC2 is epistatic to var2, and that a lack of ClpC2 does not markedly alter the composition of the thylakoid membrane. Suppression by clpC2 is not allele-specific. Our results suggest that clpC2 is a suppressor of thylakoid biogenesis and maintenance and that ClpC2 might act by accelerating photooxidative stress. Arabidopsis has two ClpC genes (ClpC1 and ClpC2), and mutants with down-regulated expression of both genes have a phenotype different from clpC2, suggesting that ClpC1 and ClpC2 act synergistically and͞or that they are only partially redundant. The isolation of a clpC2 mutant represents an important advance in the generation of tools to understand Hsp100 function and insight into the mechanisms of protein quality control in plants.P rotein concentrations within all compartments of the eukaryotic cell are regulated by various mechanisms of protein quality control (1, 2). Chaperones and ATP-dependent proteases are important components of these surveillance systems. In chloroplasts, protein quality control is mediated, in part, by three classes of ATP-dependent proteases that also have chaperone activities: Clp, Lon, and FtsH (3-5). The regulatory (ATPase) and proteolytic domains are located on separate subunits in Clp, whereas in FtsH and Lon they reside on the same polypeptide. These three proteases are evolutionarily derived from the prokaryotic-like ancestors of chloroplasts. A major unanswered question is how the various proteases and chaperones interact to provide an integrated network of protein quality control in the plastid.Photosynthetic organs of the Arabidopsis var2 variegation mutant have green sectors with cells that contain normalappearing chloroplasts and white sectors with cells that have abnormal plastids lacking organized lamellar structures (6, 7). This mutant defines a nuclear recessive gene for a plastidlocalized FtsH homolog (designated AtFtsH2) (8, 9). FtsH genes comprise small multigene families in plants, and the products of these genes are targeted to both plastids and mitochondria (10-12). Arabidopsis contains 12 AtFtsH genes, including three pairs of closely relate...

The GreenCut2 Resource, a Phylogenomically Derived Inventory of Proteins Specific to the Plant Lineage

“…Recently, the GreenCut2 signaling protein At2g48070 (CrCPLD33) was shown to mediate the chloroplast oxidative burst, which is part of the plant's immune response (76). Other types of chloroplast-nucleus signaling pathways mediated by GreenCut2 proteins might include coordination of plastid division during the cell cycle, modulation of the synthesis of thylakoid membranes (77,78), and control of the stoichiometry of photosynthetic complexes within those membranes.…”

The Balance between Protein Synthesis and Degradation in Chloroplasts Determines Leaf Variegation inArabidopsis yellow variegatedMutants

“…The observed suppression of leaf variegation by fug1 and clpc2 is similar in this sense. Park and Rodermel (2004) have reported that mutation or inactivation of ClpC2, a subunit comprising an ATPase complex of the Clp protease, leads to a suppression of variegation in var2. Loss of ClpC2 itself does not cause any visible phenotype, whereas loss of another isomer ClpC1 results in the decrease of Fv/Fm and a chlorotic phenotype (Sjö gren et al, 2004;Clarke et al, 2005;Kovacheva et al, 2005).…”

“…Indeed, the screening of var2 suppressors has been successfully performed by Park and Rodermel (2004), suggesting that several recessive mutations exist. In this study, we focused on one of the var2 suppressors that was isolated by our forward genetics screen.…”

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

“…Similarly, in otp70, the splicing of rpoC1 is compromised, which also leads to abnormal plastidencoded plastid RNA polymerase activities (18). In addition to these two categories of genes, virescent mutants have also been observed at rather high frequency in our screens for yellow variegated (var2) suppressors (19). One such suppressor line, ems2505, defines a leaky allele of an essential gene coding for a novel chloroplast protein that is indispensable for chloroplast development and plant survival (20).…”