Sub‐cellular location of FtsH proteases in the cyanobacterium Synechocystis sp. PCC 6803 suggests localised PSII repair zones in the thylakoid membranes

In cyanobacteria and chloroplasts, exposure to HL damages the photosynthetic apparatus, especially the D1 subunit of Photosystem II. To avoid chronic photoinhibition, a PSII repair cycle operates to replace damaged PSII subunits with newly synthesised versions. To determine the sub‐cellular location of this process, we examined the localisation of FtsH metalloproteases, some of which are directly involved in degrading damaged D1. We generated transformants of the cyanobacterium Synechocystis sp. PCC6803 expressing GFP‐tagged versions of its four FtsH proteases. The ftsH2–gfp strain was functional for PSII repair under our conditions. Confocal microscopy shows that FtsH1 is mainly in the cytoplasmic membrane, while the remaining FtsH proteins are in patches either in the thylakoid or at the interface between the thylakoid and cytoplasmic membranes. HL exposure which increases the activity of the Photosystem II repair cycle led to no detectable changes in FtsH distribution, with the FtsH2 protease involved in D1 degradation retaining its patchy distribution in the thylakoid membrane. We discuss the possibility that the FtsH2–GFP patches represent Photosystem II ‘repair zones’ within the thylakoid membranes, and the possible advantages of such functionally specialised membrane zones. Anti‐GFP affinity pull‐downs provide the first indication of the composition of the putative repair zones.     

Early emergence of the FtsH proteases involved in photosystem II repair

Efficient degradation of damaged D1 during the repair of PSII is carried out by a set of dedicated FtsH proteases in the thylakoid membrane. Here we investigated whether the evolution of FtsH could hold clues to the origin of oxygenic photosynthesis. A phylogenetic analysis of over 6000 FtsH protease sequences revealed that there are three major groups of FtsH proteases originating from gene duplication events in the last common ancestor of bacteria, and that the FtsH proteases involved in PSII repair form a distinct clade branching out before the divergence of FtsH proteases found in all groups of anoxygenic phototrophic bacteria. Furthermore, we showed that the phylogenetic tree of FtsH proteases in phototrophic bacteria is similar to that for Type I and Type II reaction centre proteins. We conclude that the phylogeny of FtsH proteases is consistent with an early origin of photosynthetic water oxidation chemistry.     

Involvement of the HtrA family of proteases in the protection of the cyanobacterium Synechocystis PCC 6803 from light stress and in the repair of photosystem II

Photosystem II (PSII) is prone to irreversible light-induced damage, with the D1 polypeptide a major target. Repair processes operate in the cell to replace a damaged D1 subunit within the complex with a newly synthesized copy. As yet, the molecular details of PSII repair are relatively obscure despite the critical importance of this process for maintaining PSII activity and cell viability. We are using the cyanobacterium Synechocystis sp. PCC 6803 to identify the various proteases and chaperones involved in D1 turnover in vivo. Two families of proteases are being studied: the FtsH family (four members) of Zn(2+)-activated nucleotide-dependent proteases; and the HtrA (or DegP) family (three members) of serine-type proteases. In this paper, we report the results of our studies on a triple mutant in which all three copies of the htrA gene family have been inactivated. Growth of the mutant on agar plates was inhibited at high light intensities, especially in the presence of glucose. Oxygen evolution measurements indicated that, under conditions of high light, the rate of synthesis of functional PSII was less in the mutant than in the wild-type. Immunoblotting experiments conducted on cells blocked in protein synthesis further indicated that degradation of D1 was slowed in the mutant. Overall, our observations indicate that the HtrA family of proteases are involved in the resistance of Synechocystis 6803 to light stress and play a part, either directly or indirectly, in the repair of PSII in vivo.     

FtsH-mediated repair of the photosystem II complex in response to light stress

A common feature of light stress in plants, algae, and cyanobacteria is the light-induced damage to the photosystem II complex (PSII), which catalyses the photosynthetic oxidation of water to molecular oxygen. A repair cycle operates to replace damaged subunits within PSII, in particular, the D1 reaction centre polypeptide, by newly synthesized copies. As yet the molecular details of this physiologically important process remain obscure. A key aspect of the process that has attracted much attention is the identity of the protease or proteases involved in D1 degradation. The results are summarized here of recent mutagenesis experiments that were designed to assess the functional importance of the DegP/HtrA and FtsH protease families in the cyanobacterium Synechocystis sp. PCC 6803. Based on these results and the analysis of Arabidopsis mutants, a general model for PSII repair is suggested in which FtsH complexes alone are able to degrade damaged D1.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a DeltaFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in DeltaDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in DeltaFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the DeltaDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

Role of FtsH2 in the repair of Photosystem II in mutants of the cyanobacterium Synechocystis PCC 6803 with impaired assembly or stability of the CaMn4 cluster

The FtsH2 protease, encoded by the slr0228 gene, plays a key role in the selective degradation of photodamaged D1 protein during the repair of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. To test whether additional proteases might be involved in D1 degradation during high rates of photodamage, we have studied the synthesis and degradation of the D1 protein in ΔPsbO and ΔPsbV mutants, in which the CaMn₄ cluster catalyzing oxygen evolution is less stable, and in the D1 processing mutants, D1-S345P and ΔCtpA, which are unable to assemble a functional cluster. All four mutants exhibited a dramatically increased rate of D1 degradation in high light compared to the wild-type. Additional inactivation of the ftsH2 gene slowed the rate of D1 degradation dramatically and increased the level of PSII complexes. We conclude that FtsH2 plays a major role in the degradation of both precursor and mature forms of D1 following donor-side photoinhibition. However, this conclusion concerned only D1 assembled into larger complexes containing at least D2 and CP47. In the ΔpsbEFLJ deletion mutant blocked at an early stage in PSII assembly, unassembled D1 protein was efficiently degraded in the absence of FtsH2 pointing to the involvement of other protease(s). Significantly, the ΔPsbO mutant displayed unusually low levels of cellular chlorophyll at extremely low-light intensities. The possibilities that PSII repair may limit the availability of chlorophyll for the biogenesis of other chlorophyll-binding proteins and that PsbO might have a regulatory role in PSII repair are discussed.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

Recent advances in understanding the assembly and repair of photosystem II

Background

Photosystem II (PSII) is the light-driven water:plastoquinone oxidoreductase of oxygenic photosynthesis and is found in the thylakoid membrane of chloroplasts and cyanobacteria. Considerable attention is focused on how PSII is assembled in vivo and how it is repaired following irreversible damage by visible light (so-called photoinhibition). Understanding these processes might lead to the development of plants with improved growth characteristics especially under conditions of abiotic stress.

Scope

Here we summarize recent results on the assembly and repair of PSII in cyanobacteria, which are excellent model organisms to study higher plant photosynthesis.

Conclusions

Assembly of PSII is highly co-ordinated and proceeds through a number of distinct assembly intermediates. Associated with these assembly complexes are proteins that are not found in the final functional PSII complex. Structural information and possible functions are beginning to emerge for several of these ‘assembly’ factors, notably Ycf48/Hcf136, Psb27 and Psb28. A number of other auxiliary proteins have been identified that appear to have evolved since the divergence of chloroplasts and cyanobacteria. The repair of PSII involves partial disassembly of the damaged complex, the selective replacement of the damaged sub-unit (predominantly the D1 sub-unit) by a newly synthesized copy, and reassembly. It is likely that chlorophyll released during the repair process is temporarily stored by small CAB-like proteins (SCPs). A model is proposed in which damaged D1 is removed in Synechocystis sp. PCC 6803 by a hetero-oligomeric complex composed of two different types of FtsH sub-unit (FtsH2 and FtsH3), with degradation proceeding from the N-terminus of D1 in a highly processive reaction. It is postulated that a similar mechanism of D1 degradation also operates in chloroplasts. Deg proteases are not required for D1 degradation in Synechocystis 6803 but members of this protease family might play a supplementary role in D1 degradation in chloroplasts under extreme conditions.     

The exposed N-terminal tail of the D1 subunit is required for rapid D1 degradation during photosystem II repair in Synechocystis sp PCC 6803

The selective replacement of photodamaged D1 protein within the multisubunit photosystem II (PSII) complex is an important photoprotective mechanism in chloroplasts and cyanobacteria. FtsH proteases are involved at an early stage of D1 degradation, but it remains unclear how the damaged D1 subunit is recognized, degraded, and replaced. To test the role of the N-terminal region of D1 in PSII biogenesis and repair, we have constructed mutants of the cyanobacterium Synechocystis sp PCC 6803 that are truncated at the exposed N terminus. Removal of 5 or 10 residues blocked D1 synthesis, as assessed in radiolabeling experiments, whereas removal of 20 residues restored the ability to assemble oxygen-evolving dimeric PSII complexes but inhibited PSII repair at the level of D1 degradation. Overall, our results identify an important physiological role for the exposed N-terminal tail of D1 at an early step in selective D1 degradation. This finding has important implications for the recognition of damaged D1 and its synchronized replacement by a newly synthesized subunit.     

Coordinated regulation and complex formation of yellow variegated1 and yellow variegated2, chloroplastic FtsH metalloproteases involved in the repair cycle of photosystem II in Arabidopsis thylakoid membranes

Arabidopsis yellow variegated1 (VAR1) and VAR2 are separate loci that encode similar chloroplast FtsH proteases. To date, FtsH is the best-characterized protease in thylakoid membranes involved in the turnover of photosynthetic protein complexes. It comprises a protein family that is encoded by 12 different nuclear genes in Arabidopsis. We show here that nine FtsH proteins are located in the chloroplasts. Mutations in either VAR1 or VAR2 cause typical leaf variegation and sensitivity to photoinhibition. By contrast, none of these phenotypes was observed in T-DNA insertion mutants in other ftsH genes (ftsh1, ftsh6, and ftsh8) closely related to VAR1 and VAR2. This finding suggests that VAR1 and VAR2 play a predominant role in the photosystem II repair cycle in thylakoid membranes. By generating VAR1- and VAR2-specific antibodies, we found that loss of either VAR1 or VAR2 results in the decreased accumulation of the other. Thus, the genetic nonredundancy between VAR1 and VAR2 could be attributed to their coordinated regulation at the protein level. These observations led us to examine whether VAR1 and VAR2 form a complex. Sucrose density gradient and gel filtration analyses revealed a complex of approximately 400 to 450 kD, probably representing a hexamer. Furthermore, VAR1 and VAR2 were shown to coprecipitate by immunoprecipitation using VAR1- and VAR2-specific antibodies. The majority of VAR1 appears to exist as heterocomplexes with VAR2, whereas VAR2 may be present as homocomplexes as well. Based on these results, we conclude that VAR1 and VAR2 are the major components of an FtsH complex involved in the repair of photodamaged proteins in thylakoid membranes.     

Possible compensatory role among chloroplast proteases under excess-light stress condition

The reaction center protein D1 of photosystem II (PSII), known as a primary target of photodamage, is repaired efficiently by the PSII repair cycle, to cope with constant photooxidative damage. Recent studies of Arabidopsis show that the endo-type Deg protease and the exo-type FtsH proteases cooperatively degrade D1 in the PSII repair in vivo. It is particularly interesting that we observed upregulation of Clp and SppA proteases when FtsH was limited in the mutant lacking FtsH2. To examine how the complementary functions of chloroplastic proteases are commonly regulated, we undertook a high-light stress on wild-type Arabidopsis leaves. The result that wild type leaves also showed increased levels of these proteases upon exposure to excessively strong illumination not only revealed the importance of FtsH and Deg in the PSII repair, but also implied cooperation among chloroplastic proteases under chronic stress conditions.     

The deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle

Members of the DegP/HtrA (or Deg) family of proteases are found widely in nature and play an important role in the proteolysis of misfolded and damaged proteins. As yet, their physiological role in oxygenic photosynthetic organisms is unclear, although it has been widely speculated that they participate in the degradation of the photodamaged D1 subunit in the photosystem two complex (PSII) repair cycle, which is needed to maintain PSII activity in both cyanobacteria and chloroplasts. We have examined the role of the three Deg proteases found in the cyanobacterium Synechocystis sp. PCC 6803 through analysis of double and triple insertion mutants. We have discovered that these proteases show overlap in function and are involved in a number of key physiological responses ranging from protection against light and heat stresses to phototaxis. In previous work, we concluded that the Deg proteases played either a direct or an indirect role in PSII repair in a glucose-tolerant version of Synechocystis 6803 (Silva, P., Choi, Y. J., Hassan, H. A., and Nixon, P. J. (2002) Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 1461-1467). In this work, we have now been able to demonstrate unambiguously, using a triple deg mutant created in the wild type strain of Synechocystis 6803, that the Deg proteases are not obligatory for PSII repair and D1 degradation. We therefore conclude that although the Deg proteases are needed for photoprotection of Synechocystis sp. PCC 6803, they do not play an essential role in D1 turnover and PSII repair in vivo.     

Ammonia triggers photodamage of photosystem II in the cyanobacterium Synechocystis sp. strain PCC 6803

Ammonia has long been known to be toxic for many photosynthetic organisms; however, the target for its toxicity remains elusive. Here, we show that in the cyanobacterium Synechocystis sp. strain PCC 6803, ammonia triggers a rapid photodamage of photosystem II (PSII). Whereas wild-type cells can cope with this damage by turning on the FtsH2-dependent PSII repair cycle, the FtsH2-deficient mutant is highly sensitive and loses PSII activity at millimolar concentration of ammonia. Ammonia-triggered PSII destruction is light dependent and occurs already at low photon fluence rates. Experiments with monochromatic light showed that ammonia-promoted PSII photoinhibition is executed by wavebands known to directly destroy the manganese cluster in the PSII oxygen-evolving complex, suggesting that the oxygen-evolving complex may be a direct target for ammonia toxicity.     

Cooperative D1 Degradation in the Photosystem II Repair Mediated by Chloroplastic Proteases in Arabidopsis

Light energy constantly damages photosynthetic apparatuses, ultimately causing impaired growth. Particularly, the sessile nature of higher plants has allowed chloroplasts to develop unique mechanisms to alleviate the irreversible inactivation of photosynthesis. Photosystem II (PSII) is known as a primary target of photodamage. Photosynthetic organisms have evolved the so-called PSII repair cycle, in which a reaction center protein, D1, is degraded rapidly in a specific manner. Two proteases that perform processive or endopeptidic degradation, FtsH and Deg, respectively, participate in this cycle. To examine the cooperative D1 degradation by these proteases, we engaged Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) and Deg5/Deg8 (deg5 deg8) in detecting D1 cleaved fragments. We detected several D1 fragments only under the var2 background, using amino-terminal or carboxyl-terminal specific antibodies of D1. The appearance of these D1 fragments was inhibited by a serine protease inhibitor and by deg5 deg8 mutations. Given the localization of Deg5/Deg8 on the luminal side of thylakoid membranes, we inferred that Deg5/Deg8 cleaves D1 at its luminal loop connecting the transmembrane helices C and D and that the cleaved products of D1 are the substrate for FtsH. These D1 fragments detected in var2 were associated with the PSII monomer, dimer, and partial disassembly complex but not with PSII supercomplexes. It is particularly interesting that another processive protease, Clp, was up-regulated and appeared to be recruited from stroma to the thylakoid membrane in var2, suggesting compensation for FtsH deficiency. Together, our data demonstrate in vivo cooperative degradation of D1, in which Deg cleavage assists FtsH processive degradation under photoinhibitory conditions.     

Phosphorylation of photosystem II core proteins prevents undesirable cleavage of D1 and contributes to the fine‐tuned repair of photosystem II

Photosystem II (PSII) is a primary target for light‐induced damage in photosynthetic protein complexes. To avoid photoinhibition, chloroplasts have evolved a repair cycle with efficient degradation of the PSII reaction center protein, D1, by the proteases FtsH and Deg. Earlier reports have described that phosphorylated D1 is a poor substrate for proteolysis, suggesting a mechanistic role for protein phosphorylation in PSII quality control, but its precise role remains elusive. STN8, a protein kinase, plays a central role in this phosphorylation process. To elucidate the relationship between phosphorylation of D1 and the protease function we assessed in this study the involvement of STN8, using Arabidopsis thaliana mutants lacking FtsH2 [yellow variegated2 (var2)] and Deg5/Deg8 (deg5 deg8). In support of our presumption we found that phosphorylation of D1 increased more in var2. Furthermore, the coexistence of var2 and stn8 was shown to recover the delay in degradation of D1, resulting in mitigation of the high vulnerability to photoinhibition of var2. Partial D1 cleavage fragments that depended on Deg proteases tended to increase, with concomitant accumulation of reactive oxygen species in the mutants lacking STN8. We inferred that the accelerated degradation of D1 in var2 stn8 presents a tradeoff in that it improved the repair of PSII but simultaneously enhanced oxidative stress. Together, these results suggest that PSII core phosphorylation prevents undesirable cleavage of D1 by Deg proteases, which causes cytotoxicity, thereby balancing efficient linear electron flow and photo‐oxidative damage. We propose that PSII core phosphorylation contributes to fine‐tuned degradation of D1.     

Chloroplast proteases: possible regulators of gene expression?

A wide range of proteolytic processes in the chloroplast are well recognized. These include processing of precursor proteins, removal of oxidatively damaged proteins, degradation of proteins missing their prosthetic groups or their partner subunit in a protein complex, and adjustment of the quantity of certain chloroplast proteins in response to changing environmental conditions. To date, several chloroplast proteases have been identified and cloned. The chloroplast processing enzyme is responsible for removing the transit peptides of newly imported proteins. The thylakoid processing peptidase removes the thylakoid-transfer domain from proteins translocated into the thylakoid lumen. Within the lumen, Tsp removes the carboxy-terminal tail of the precursor of the PSII D1 protein. In contrast to these processing peptidases which perform a single endo-proteolytic cut, processive proteases that can completely degrade substrate proteins also exist in chloroplasts. The serine ATP-dependent Clp protease, composed of the proteolytic subunit ClpP and the regulatory subunit ClpC, is located in the stroma, and is involved in the degradation of abnormal soluble and membrane-bound proteins. The ATP-dependent metalloprotease FtsH is bound to the thylakoid membrane, facing the stroma. It degrades unassembled proteins and is involved in the degradation of the D1 protein of PSII following photoinhibition. DegP is a serine protease bound to the lumenal side of the thylakoid membrane that might be involved in the chloroplast response to heat. All these peptidases and proteases are homologues of known bacterial enzymes. Since ATP-dependent bacterial proteases and their mitochondrial homologues are also involved in the regulation of gene expression, via their determining the levels of key regulatory proteins, chloroplast proteases are expected to play a similar role.     

The FtsH protease slr0228 is important for quality control of photosystem II in the thylakoid membrane of Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 contains four members of the FtsH protease family. One of these, FtsH (slr0228), has been implicated recently in the repair of photodamaged photosystem II (PSII) complexes. We have demonstrated here, using a combination of blue native PAGE, radiolabeling, and immunoblotting, that FtsH (slr0228) is required for selective replacement of the D1 reaction center subunit in both wild type PSII complexes and in PSII subcomplexes lacking the PSII chlorophyll a-binding subunit CP43. To test whether FtsH (slr0228) has a more general role in protein quality control in vivo, we have studied the synthesis and degradation of PSII subunits in wild type and in defined insertion and missense mutants incapable of proper assembly of the PSII holoenzyme. We discovered that, when the gene encoding FtsH (slr0228) was disrupted in these strains, the overall level of assembly intermediates and unassembled PSII proteins markedly increased. Pulse-chase experiments showed that this was due to reduced rates of degradation in vivo. Importantly, analysis of epitope-tagged and green fluorescent protein-tagged strains revealed that slr0228 was present in the thylakoid and not the cytoplasmic membrane. Overall, our results show that FtsH (slr0228) plays an important role in controlling the removal of PSII subunits from the thylakoid membrane and is not restricted to selective D1 turnover.     

Revised scheme for the mechanism of photoinhibition and its application to enhance the abiotic stress tolerance of the photosynthetic machinery

When photosynthetic organisms are exposed to abiotic stress, their photosynthetic activity is significantly depressed. In particular, photosystem II (PSII) in the photosynthetic machinery is readily inactivated under strong light and this phenomenon is referred to as photoinhibition of PSII. Other types of abiotic stress act synergistically with light stress to accelerate photoinhibition. Recent studies of photoinhibition have revealed that light stress damages PSII directly, whereas other abiotic stresses act exclusively to inhibit the repair of PSII after light-induced damage (photodamage). Such inhibition of repair is associated with suppression, by reactive oxygen species (ROS), of the synthesis of proteins de novo and, in particular, of the D1 protein, and also with the reduced efficiency of repair under stress conditions. Gene-technological improvements in the tolerance of photosynthetic organisms to various abiotic stresses have been achieved via protection of the repair system from ROS and, also, by enhancing the efficiency of repair via facilitation of the turnover of the D1 protein in PSII. In this review, we summarize the current status of research on photoinhibition as it relates to the effects of abiotic stress and we discuss successful strategies that enhance the activity of the repair machinery. In addition, we propose several potential methods for activating the repair system by gene-technological methods.     

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)–tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2⁻ mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.     

The thylakoid lumen protease Deg1 is involved in the repair of photosystem II from photoinhibition in Arabidopsis

Deg1 is a Ser protease peripherally attached to the lumenal side of the thylakoid membrane. Its physiological function is unknown, but its localization makes it a suitable candidate for participation in photoinhibition repair by degradation of the photosystem II reaction center protein D1. We transformed Arabidopsis thaliana with an RNA interference construct and obtained plants with reduced levels of Deg1. These plants were smaller than wild-type plants, flowered earlier, were more sensitive to photoinhibition, and accumulated more of the D1 protein, probably in an inactive form. Two C-terminal degradation products of the D1 protein, of 16 and 5.2 kD, accumulated at lower levels compared with the wild type. Moreover, addition of recombinant Deg1 to inside-out thylakoid membranes isolated from the mutant could induce the formation of the 5.2-kD D1 C-terminal fragment, whereas the unrelated proteases trypsin and thermolysin could not. Immunoblot analysis revealed that mutants containing less Deg1 also contain less FtsH protease, and FtsH mutants contain less Deg1. These results suggest that Deg1 cooperates with the stroma-exposed proteases FtsH and Deg2 in degrading D1 protein during repair from photoinhibition by cleaving lumen-exposed regions of the protein. In addition, they suggest that accumulation of Deg1 and FtsH proteases may be coordinated.