The family of Deg proteases in cyanobacteria and chloroplasts of higher plants

The family of Deg proteases is present in nearly all organisms from bacteria to higher plants. This family consists of ATP-independent serine endopeptidases with a catalytic domain of trypsin type and up to three PDZ domains, involved in protein–protein interactions. Sixteen deg genes (originally named deg P1–16) were found in Arabidopsis thaliana , and the chloroplast location was predicted or experimentally proven for seven proteins. The cyanobacterium Synechocystis sp. PCC6803 contains three Deg homologues, HtrA (DegP), HhoA (DegQ) and HhoB (DegS), but their number can vary between one and six in other photosynthetic Prokaryota. Interestingly, all of these proteases are evolutionarily more closely related within one species than proteases with the same names present in other organisms. This means that Deg proteases from A. thaliana are not necessarily the closest relatives of cyanobacterial DegP. Therefore, we propose to change the misleading original name 'DegP' to 'Deg' for A. thaliana enzymes. Here, we summarize the expression, location and functions of Deg proteases from cyanobacteria and chloroplasts of higher plants, with special emphasis on their role in the photosystem II (PSII) repair cycle under light 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.     

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.     

High light stimulates Deg1-dependent cleavage of the minor LHCII antenna proteins CP26 and CP29 and the PsbS protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The chloroplast Deg1 protein performs proteolytic cleavage of the photodamaged D1 protein of the photosystem II (PSII) reaction center, PSII extrinsic subunit PsbO and the soluble electron carrier plastocyanin. Using biochemical, immunological and mass spectrometry approaches we showed that the heterogeneously expressed Deg1 protease from Arabidopsis thaliana can be responsible for the degradation of the monomeric light-harvesting complex antenna subunits of PSII (LHCII), CP26 and CP29, as well as PSII-associated PsbS (CP22/NPQ4) protein. The results may indicate that cytochrome b ₆ protein and two previously unknown thylakoid proteins, Ptac16 and an 18.3-kDa protein, may be the substrates for Deg1. The interaction of Deg1 with the PsbS protein and the minor LHCII subunits implies its involvement in the regulation of both excess energy dissipation and state transition adaptation processes.     

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.     

Assembling and maintaining the Photosystem II complex in chloroplasts and cyanobacteria

Plants, algae and cyanobacteria grow because of their ability to use sunlight to extract electrons from water. This vital reaction is catalysed by the Photosystem II (PSII) complex, a large multi-subunit pigment-protein complex embedded in the thylakoid membrane. Recent results show that assembly of PSII occurs in a step-wise fashion in defined regions of the membrane system, involves conserved auxiliary factors and is closely coupled to chlorophyll biosynthesis. PSII is also repaired following damage by light. FtsH proteases play an important role in selectively removing damaged proteins from the complex, both in chloroplasts and cyanobacteria, whilst undamaged subunits and pigments are recycled. The chloroplastic Deg proteases play a supplementary role in PSII repair.     

A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II

Although light is the ultimate substrate in photosynthesis, it can also be harmful and lead to oxidative damage of the photosynthetic apparatus. The main target for light stress is the central oxygen-evolving photosystem II (PSII) and its D1 reaction centre protein. Degradation of the damaged D1 protein and its rapid replacement by a de novo synthesized copy represent the important repair mechanism of PSII crucial for plant survival under light stress conditions. Here we report the isolation of a single-copy nuclear gene from Arabidopsis thaliana, encoding a protease that performs GTP-dependent primary cleavage of the photodamaged D1 protein and hence catalysing the key step in the repair cycle in plants. This protease, designated DegP2, is a homologue of the prokaryotic Deg/Htr family of serine endopeptidases and is associated with the stromal side of the non-appressed region of the thylakoid membranes. Increased expression of DegP2 under high salt, desiccation and light stress conditions was measured at the protein level.     

The thylakoid protease Deg1 is involved in photosystem‐II assembly in Arabidopsis thaliana

DegP proteases have been shown to possess both chaperone and protease activities. The proteolytic activities of chloroplast DegP‐like proteases have been well documented. However, whether chloroplast Deg proteases also have chaperone activities has remained unknown. Here we show that chloroplast Deg1 also has chaperone activities, like its Escherichia coli ortholog DegP. Transgenic plants with reduced levels of Deg1 accumulated normal levels of different subunits of the major photosynthetic protein complexes, but their levels of photosystem‐II (PSII) dimers and supercomplexes were reduced. In vivo pulse‐chase protein labeling experiments showed that the assembly of newly synthesized proteins into PSII dimers and supercomplexes was impaired, although the synthesis rate of chloroplast proteins was unaffected in the transgenic lines. Protein overlay assays provided direct evidence that Deg1 interacts with the PSII reaction center protein D2. These results suggest that Deg1 assists the assembly of the PSII complex, probably through interaction with the PSII reaction center D2 protein.     

Proteolytic system of plant mitochondria

The existence of a proteolytic system which can specifically recognize and cleave proteins in mitochondria is now well established. The components of this system comprise processing peptidases, ATP-dependent peptidases and oligopeptidases. A short overview of experimentally confirmed proteases mainly from Arabidopsis thaliana is provided. The role of the mitochondrial peptidases in plant growth and development is emphasized. We also discuss the possibility of existence of as yet unidentified plant homologs of yeast mitochondrial ATP-independent proteases.     

Molecular biology of intracellular protein trafficking

The evidence accumulated to date indicates that protein compartmentalization is mediated through specific regions of proteins destined for translocation into subcellular organelles. Proteins targeted to mitochondria, chloroplasts or the endoplasmic reticulum have 'transit' sequences contained in amino-terminal peptide extensions. However, most peroxisomal proteins do not have amino-terminal extensions. Protein importation into mitochondria has been extensively studied and characterized. This post-translational process appears to involve receptors on the mitochondrial outer membrane, and is dependent upon the electrochemical gradient across the inner membrane. Translocation to one of the submitochondrial compartments is determined by the type of transit sequence contained in a mitochondrial protein. The majority of imported mitochondrial proteins are proteolytically altered prior to assembly into oligomeric enzyme complexes. Protein importation into peroxisomes is distinctly different from importation into mitochondria. Although both processes are post-translational, their only other similarity is a requirement for ATP. In this review, we present and compare recent evidence for both mitochondrial and peroxisomal protein importation.     

Molecular chaperones and the biogenesis of mitochondria and peroxisomes

Summary— A review of the proteinaceous machinery involved in protein sorting pathways and protein folding and assembly in mitochondria and peroxisomes is presented. After considering the various sorting pathways and targeting signals of mitochondrial and peroxisomal proteins, we make a comparative dissection of the protein factors involved in: i) the stabilization of cytosolic precursor proteins in a translocation competent conformation; ii) the membrane import apparatus of mitochondria and peroxisomes; iii) the processing of mitochondrial precursor proteins, and the eventual processing of certain peroxisomal precursor, in the interior of the organelles; and iv) the requirement of molecular chaperones for appropriate folding and assembly of imported proteins in the matrix of both organelles. Those aspects of mitochondrial biogenesis that have developed rapidly during the last few years, such as the requirement of molecular chaperones, are stressed in order to stimulate further parallel investigations aimed to understand the origin, biochemistry, molecular biology and pathology of peroxisomes. In this regard, a brief review of findings from our group and others is presented in which the role of the F₁-ATPase α-subunit is pointed out as a molecular chaperone of mitochondria and chloroplasts. In addition, data are presented that could question our previous indication that the immunoreactive protein found in the rat liver peroxisomes is due to the presence of the F₁-ATPase α-subunit.     

Plant mitochondrial rhomboid, AtRBL12, has different substrate specificity from its yeast counterpart

Rhomboid proteins comprise a class of serine proteases that are conserved in all kingdoms of organisms. They contain six or seven transmembrane helices and control a wide range of cellular functions and developmental processes by intramembrane proteolysis. This paper provides experimental evidence for the existence of rhomboid proteases in plant mitochondria and chloroplasts. Among 15 putative rhomboid-like proteins in Arabidopsis thaliana, we selected five predicted as mitochondrially targeted. For these proteins we performed the GFP transient assay, and identified two homologues, AtRBL11 (At5g25752) and AtRBL12 (At1g18600) to be targeted into plastids and mitochondria, respectively. Phylogenetic analysis reveals that AtRBL12 or AtRBL11 have only one clear orthologue in plant species with completely sequenced genomes. Complementation of the yeast lacking a functional copy of mitochondrial rhomboid with AtRBL12 indicates that this plant protease, in contrast to the human orthologue, does not recognize the yeast substrates, cytochrome c peroxidase (Ccp1) or dynamin-like GTPase (Mgm1). In agreement with this, we did not observe processing of Mgm1 when labeled precursor of this protein was incubated in vitro with Arabidopsis mitochondrial extract. Our results imply that plant mitochondrial rhomboids function in a specific manner and thus differ from their yeast and mammal counterparts.     

The family of Deg/HtrA proteases: from Escherichia coli to Arabidopsis

In the genomic era, an increasing number of protease genes have been identified in various organisms. During the last few years, many of these proteases have been characterized using biochemical as well as molecular biological techniques. However, neither the precise location nor the physiological substrates of these enzymes has been identified in many cases, including the Deg/HtrA proteases, a family of serine-type ATP-independent proteases. This family has become especially interesting for many researchers following the determination of the crystal structures of an Escherichia coli and a human Deg/HtrA protease. A breakthrough in photosynthesis research has revealed that a Deg/HtrA protease of Arabidopsis thaliana is involved in the degradation of the D1 protein of photosystem II following photoinhibition. In this review, the available data on Deg/HtrAs of different organisms are compared with those from the photoautotroph cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana .     

Chloroplast and mitochondrial proteases in Arabidopsis. A proposed nomenclature

The identity and scope of chloroplast and mitochondrial proteases in higher plants has only started to become apparent in recent years. Biochemical and molecular studies suggested the existence of Clp, FtsH, and DegP proteases in chloroplasts, and a Lon protease in mitochondria, although currently the full extent of their role in organellar biogenesis and function remains poorly understood. Rapidly accumulating DNA sequence data, especially from Arabidopsis, has revealed that these proteolytic enzymes are found in plant cells in multiple isomeric forms. As a consequence, a systematic approach was taken to catalog all these isomers, to predict their intracellular location and putative processing sites, and to propose a standard nomenclature to avoid confusion and facilitate scientific communication. For the Clp protease most of the ClpP isomers are found in chloroplasts, whereas one is mitochondrial. Of the ATPase subunits, the one ClpD and two ClpC isomers are located in chloroplasts, whereas both ClpX isomers are present in mitochondria. Isomers of the Lon protease are predicted in both compartments, as are the different forms of FtsH protease. DegP, the least characterized protease in plant cells, has the most number of isomers and they are predicted to localize in several cell compartments. These predictions, along with the proposed nomenclature, will serve as a framework for future studies of all four families of proteases and their individual isomers.     

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.     

How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach

Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.     

A yeast mitochondrial leader peptide functions in vivo as a dual targeting signal for both chloroplasts and mitochondria.

A fusion protein was expressed in transgenic tobacco and yeast cells to examine the functional conservation of mechanisms for importing precursor proteins from the cytosol into mitochondria and chloroplasts. The test protein consisted of the mitochondrial leader peptide from the yeast precursor to cytochrome oxidase subunit Va (prC5) fused to the reporter protein chloramphenicol acetyltransferase. This protein, denoted prC5/CAT, was transported into the mitochondrial interior in yeast and tobacco cells. In both organisms, the mitochondrial form of prC5/CAT was smaller than the primary translation product, suggesting that proteolytic processing occurred during the transport process. prC5/CAT also was translocated into chloroplasts in vivo, accumulating to approximately the same levels as in plant mitochondria. However, accumulation of prC5/CAT in chloroplasts relative to mitochondria varied with the conditions under which plants were grown. The chloroplast form of prC5/CAT also appeared to have been proteolytically processed, yielding a mature protein of the same apparent size as that seen in mitochondria of either tobacco or yeast. Chloramphenicol acetyltransferase lacking a mitochondrial targeting peptide did not associate with either chloroplasts or mitochondria. The results demonstrated that in plant cells a single leader peptide can interact functionally with the protein translocation systems of both chloroplasts and mitochondria, and raised the possibility that certain native proteins might be shared between these two organelles.     

Processing, targeting and import into the mitochondria and peroxisomes of proteins coded by nuclear genes]

I have herein discussed two gene-enzyme families in maize whose protein products participate to purge toxic oxidants from cells, and are thus of importance to all aerobic organisms. We have demonstrated that plant mitochondria import precursor proteins (i.e., preSOD-3) in a manner analogous to other eukaryotic cells. The "transit peptide" (TP) of preSOD-3 is 31 amino acid long and has similar properties to other reported TPs for mitochondrial and chloroplastic proteins. Import to peroxisomes is uniquely different from that for mitochondria and chloroplasts in that no consensus presequence seems to be involved. Instead, targeting signals seem to be integral parts of peroxisomal proteins.     

Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different

Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F(1)beta pre-sequence, peptides derived from the F(1)beta pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F(1)beta pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P(1) position and small, uncharged amino acids or serine residues in the P'(1) position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the alpha-helical elements of the F(1)beta pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic alpha-helix and positively charged amino acid residues and degraded the F(1)beta pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F(1)beta pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.