Chloroplast Proteases

The chloroplast within the plant cell has a dynamic environment where proteases play an important role in processing of precursor proteins, degradation of incomplete proteins lacking cofactors, stress-induced degradation and removal of damaged proteins. A number of proteases in the chloroplast are well characterized and found to be localized within different compartments such as stroma, thylakoids and lumen. In recent years, an increasing number of proteases in chloroplasts have been discovered and identified as bacterial protease homologues. These include the stromal Clp, thylakoidal FtsH and lumenal DegP. The current focus is to understand their role in chloroplast regulation both at the enzyme-substrate and genetic levels.     

ATP-dependent proteases in biogenesis and maintenance of plant mitochondria

ATP-dependent proteases from three families have been identified experimentally in Arabidopsis mitochondria: four FtsH proteases (AtFtsH3, AtFtsH4, AtFtsH10, and AtFtsH11), two Lon proteases (AtLon1 and AtLon4), and one Clp protease (AtClpP2 with regulatory subunit AtClpX). In this review we discuss their submitochondrial localization, expression profiles and proposed functions, with special emphasis on their impact on plant growth and development. The best characterized plant mitochondrial ATP-dependent proteases are AtLon1 and AtFtsH4. It has been proposed that AtLon1 is necessary for proper mitochondrial biogenesis during seedling establishment, whereas AtFtsH4 is involved in maintaining mitochondrial homeostasis late in rosette development under short-day photoperiod.     

Cutting edge of chloroplast proteolysis

Chloroplasts have a dynamic protein environment and, although proteases are presumably major contributors, the identities of these crucial regulatory proteins have only recently been revealed. There are defined proteases within each of the major chloroplast compartments: the ATP-dependent Clp and FtsH proteases in the stroma and stroma-exposed thylakoid membranes, respectively, the ATP-independent DegP proteases within the thylakoid lumen and on both sides of thylakoid membranes, and the SppA protease on the stromal side of the thylakoid. All four types are homologous to proteases characterized in bacteria, but most have many isomers in higher plants. With such diversity, the challenge is to link the mode of action of each protease to the chloroplast enzymes and regulatory proteins that it targets.     

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.     

Resolving individual steps in the operation of ATP-dependent proteolytic molecular machines: from conformational changes to substrate translocation and processivity

Clp, Lon, and FtsH proteases are proteolytic molecular machines that use the free energy of ATP hydrolysis to unfold protein substrates and processively present them to protease active sites. Here we review recent biochemical and structural studies relevant to the mechanism of ATP-dependent processive proteolysis. Despite the significant structural differences among the Clp, Lon, and FtsH proteases, these enzymes share important mechanistic features. In these systems, mechanistic studies have provided evidence for ATP binding and hydrolysis-driven conformational changes that drive translocation of substrates, which has significant implications for the processive mechanism of proteolysis. These studies indicate that the nucleotide (ATP, ADP, or nonhydrolyzable ATP analogues) occupancy of the ATPase binding sites can influence the binding mode and/or binding affinity for protein substrates. A general mechanism is proposed in which the communication between ATPase active sites and protein substrate binding regions coordinates a processive cycle of substrate binding, translocation, proteolysis, and product release.     

ATP-dependent FtsH and Lon chloroplast proteases

Arabidopsis thaliana proteome contains 667 proteases; some tens of them are chloroplast-targeted proteins, encoded by genes orthologous to the ones coding for bacterial proteolytic enzymes. It is thought that chloroplast proteases are involved in chloroplasts' proteins turnover and quality control (maturation of nucleus-encoded proteins and removal of nonfunctional ones). Some ATP-dependent chloroplast proteases belonging to FtsH family (especially FtsH2 and FtsH5) are considered to be involved in numerous aspects of chloroplast and whole plant maintenance under non-stressing as well as stressing conditions. This notion is supported by severe phenotype appearance of mutants deficient in these proteases. In contrast to seemingly high physiological importance of chloroplast members of FtsH protease family, only a few individual proteins have been identified so far as their physiological targets (i.e. Lhcb1, Lhcb3, PsbA and Rieske protein). Our knowledge regarding structure and molecular mechanisms of these enzymes' action is limited when compared with what is known about FtsHs of bacterial origin. Equally limited is the knowledge about ATP-dependent Lon4 protease being the single known chloroplast-targeted ortholog of Lon protease of Escherichia coli.     

PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix

Loss-of-function mutations in PINK1, which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson''s disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, we performed a screen for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. We found that genetic perturbations targeting the matrix-localized protease Lon caused dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPR^mt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPR^mt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. Our findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria.     

Quality control of photosystem II

Photosystem II is particularly vulnerable to excess light. When illuminated with strong visible light, the reaction center D1 protein is damaged by reactive oxygen molecules or by endogenous cationic radicals generated by photochemical reactions, which is followed by proteolytic degradation of the damaged D1 protein. Homologs of prokaryotic proteases, such as ClpP, FtsH and DegP, have been identified in chloroplasts, and participation of the thylakoid-bound FtsH in the secondary degradation steps of the photodamaged D1 protein has been suggested. We found that cross-linking of the D1 protein with the D2 protein, the alpha-subunit of cytochrome b(559), and the antenna chlorophyll-binding protein CP43, occurs in parallel with the degradation of the D1 protein during the illumination of intact chloroplasts, thylakoids and photosystem II-enriched membranes. The cross-linked products are then digested by a stromal protease(s). These results indicate that the degradation of the photodamaged D1 protein proceeds through membrane-bound proteases and stromal proteases. Moreover, a 33-kDa subunit of oxygen-evolving complex (OEC), bound to the lumen side of photosystem II, regulates the formation of the cross-linked products of the D1 protein in donor-side photoinhibition of photosystem II. Thus, various proteases and protein components in different compartments in chloroplasts are implicated in the efficient turnover of the D1 protein, thus contributing to the control of the quality of photosystem II under light stress conditions.     

Lon protease promotes survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> during anaerobic glucose starvation

In Escherichia coli, Lon is an ATP-dependent protease which degrades misfolded proteins and certain rapidly-degraded regulatory proteins. Given that oxidatively damaged proteins are generally degraded rather than repaired, we anticipated that Lon deficient cells would exhibit decreased viability during aerobic, but not anaerobic, carbon starvation. We found that the opposite actually occurs. Wild-type and Lon deficient cells survived equally well under aerobic conditions, but Lon deficient cells died more rapidly than the wild-type under anaerobiosis. Aerobic induction of the Clp family of ATP-dependent proteases could explain these results, but direct quantitation of Clp protein established that its level was not affected by Lon deficiency and overexpression of Clp did not rescue the cells under anaerobic conditions. We conclude that the Lon protease supports survival during anaerobic carbon starvation by a mechanism which does not depend on Clp. Shen Luo and Megan McNeill contributed equally to this research.     

Interaction of maize Opaque-2 and the transcriptional co-activators GCN5 and ADA2, in the modulation of transcriptional activity

The FtsH proteases, also called AAA proteases, are membrane-bound ATP-dependent metalloproteases. The Arabidopsis genome contains a total of 12 FtsH-like genes. Two of them, AtFtsH4 and AtFtsH11, encode proteins with a high similarity to Yme1p, a subunit of the i-AAA complex in yeast mitochondria. Phylogenetic analysis groups the AtFtsH4, AtFtsH11 and Yme1 proteins together, with AtFtsH4 being the most similar to Yme1. Using immunological method we demonstrate here that AtFtsH4 is an exclusively mitochondrial protein while AtFtsH11 is found in both chloroplasts and mitochondria. AtFtsH4 and AtFtsH11 proteases are integral parts of the inner mitochondrial membrane and expose their catalytic sites towards the intermembrane space, same as yeast i-AAA. Database searches revealed that orthologs of AtFtsH4 and AtFtsH11 are present in both monocotyledonous and dicotyledonous plants. The two plant i-AAA proteases differ significantly in their termini: the FtsH4 proteins have a characteristic alanine stretch at the C-terminal end while FtsH11s have long N-terminal extensions. Blue-native gel electrophoresis revealed that AtFtsH4 and AtFtsH11 form at least two complexes with apparent molecular masses of about 1500 kDa. This finding implies that plants, in contrast to fungi and metazoa, have more than one complex with a topology similar to that of yeast i-AAA.     

Distinctive types of ATP-dependent Clp proteases in cyanobacteria

Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis and are thought to be ancestors to plant chloroplasts. Like chloroplasts, cyanobacteria possess a diverse array of proteolytic enzymes, with one of the most prominent being the ATP-dependent Ser-type Clp protease. The model Clp protease in Escherichia coli consists of a single ClpP proteolytic core flanked on one or both ends by a HSP100 chaperone partner. In comparison, cyanobacteria have multiple ClpP paralogs plus a ClpP variant (ClpR), which lacks the catalytic triad typical of Ser-type proteases. In this study, we reveal that two distinct soluble Clp proteases exist in the unicellular cyanobacterium Synechococcus elongatus. Each protease consists of a unique proteolytic core comprised of two separate Clp subunits, one with ClpP1 and ClpP2, the other with ClpP3 and ClpR. Each core also associates with a particular HSP100 chaperone partner, ClpC in the case of the ClpP3/R core, and ClpX for the ClpP1/P2 core. The two adaptor proteins, ClpS1 and ClpS2 also interact with the ClpC chaperone protein, likely increasing the range of protein substrates targeted by the Clp protease in cyanobacteria. We also reveal the possible existence of a third Clp protease in Synechococcus, one which associates with the internal membrane network. Altogether, we show that presence of several distinctive Clp proteases in cyanobacteria, a feature which contrasts from that in most other organisms.     

Opposing effects of DNA on proteolysis of a replication initiator

DNA replication initiation proteins (Reps) are subjected to degradation by cellular proteases. We investigated how the formation of nucleoprotein complex, involving Rep and a protease, affects Rep degradation. All known Escherichia coli AAA+ cytosolic proteases and the replication initiation protein TrfA of the broad-host-range plasmid RK2 were used. Our results revealed that DNA influences the degradation process and that the observed effects are opposite and protease specific. In the case of ClpXP and ClpYQ proteases, DNA abolishes proteolysis, while in the case of ClpAP and Lon proteases it stimulates the process. ClpX and ClpY cannot interact with DNA-bound TrfA, while the ClpAP and Lon activities are enhanced by the formation of nucleoprotein complexes involving both the protease and TrfA. Lon has to interact with TrfA before contacting DNA, or this interaction can occur with TrfA already bound to DNA. The TrfA degradation by Lon can be carried out only on DNA. The absence of Lon results with higher stability of TrfA in the cell.     

Recent advances in the study of Clp, FtsH and other proteases located in chloroplasts

Several chloroplast proteases have been characterized in recent years. The ATP-dependent chloroplast proteases Clp and FtsH stand out because they form multi-subunit complexes consisting of different gene products. Surprisingly, both green and non-green plastids appear to contain a similar soluble Clp core proteolytic complex, consisting of five ClpP proteases, their non-catalytic ClpR homologs, and two ClpS homologs that have unknown function. Analyses of single and double FtsH1, FtsH2, FtsH5 and FtsH8 mutants, and overexpression of FtsH proteins in these Arabidopsis thaliana mutants show partial redundancies within pairs of closely related FtsH thylakoid proteins. The presence of at least one member of each pair is essential for functional accumulation. Other chloroplast proteases have also been identified recently. Future challenges include the identification of substrate recognition mechanisms and elucidating the role of proteases in chloroplast biogenesis and function.     

Role of the novel metallopeptidase Mop112 and saccharolysin for the complete degradation of proteins residing in different subcompartments of mitochondria

Mitochondria harbor a conserved proteolytic system that mediates the complete degradation of organellar proteins. ATP-dependent proteases, like a Lon protease in the matrix space and m- and i-AAA proteases in the inner membrane, degrade malfolded proteins within mitochondria and thereby protect the cell against mitochondrial damage. Proteolytic breakdown products include peptides and free amino acids, which are constantly released from mitochondria. It remained unclear, however, whether the turnover of malfolded proteins involves only ATP-dependent proteases or also oligopeptidases within mitochondria. Here we describe the identification of Mop112, a novel metallopeptidase of the pitrilysin family M16 localized in the intermembrane space of yeast mitochondria. This peptidase exerts important functions for the maintenance of the respiratory competence of the cells that overlap with the i-AAA protease. Deletion of MOP112 did not affect the stability of misfolded proteins in mitochondria, but resulted in an increased release from the organelle of peptides, generated upon proteolysis of mitochondrial proteins. We find that the previously described metallopeptidase saccharolysin (or Prd1) exerts a similar function in the intermembrane space. The identification of peptides released from peptidase-deficient mitochondria by mass spectrometry indicates a dual function of Mop112 and saccharolysin: they degrade peptides generated upon proteolysis of proteins both in the intermembrane and matrix space and presequence peptides cleaved off by specific processing peptidases in both compartments. These results suggest that the turnover of mitochondrial proteins is mediated by the sequential action of ATP-dependent proteases and oligopeptidases, some of them localized in the intermembrane space.     

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.     

Deg proteases and their role in protein quality control and processing in different subcellular compartments of the plant cell

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent serine endopeptidases found in almost every organism. Database searches revealed that 16 Deg paralogues are encoded by the genome of Arabidopsis thaliana, six of which were experimentally shown to be located in chloroplasts, one in peroxisomes, one in mitochondria and one in the nucleus. Two more Deg proteases are predicted to reside in chloroplasts, five in mitochondria (one of them with a dual chloroplastidial/mitochondrial localization) and the subcellular location of one protein is uncertain. This review summarizes the current knowledge on the role of Deg proteases in maintaining protein homeostasis and protein processing in various subcompartments of the plant cell. The chloroplast Deg proteases are the best examined so far, especially with respect to their role in the degradation of photodamaged photosynthetic proteins and in biogenesis of photosystem II (PSII). A combined action of thylakoid lumen and stroma Deg proteases in the primary cleavage of photodamaged D1 protein from PSII reaction centre is discussed on the basis of a recently resolved crystal structure of plant Deg1. The peroxisomal Deg protease is a processing enzyme responsible for the cleavage of N-terminal peroxisomal targeting signals (PTSs). A. thaliana mutants lacking this enzyme show reduced peroxisomal β-oxidation, indicating for the first time the impact of protein processing on peroxisomal functions in plants. Much less data is available for mitochondrial and nuclear Deg proteases. Based on the available expression data we hypothesize a role in general protein quality control and during acquired heat resistance.     

The ATP-dependent Clp protease in chloroplasts of higher plants

The best-known proteases in plastids are those that belong to families common to eubacteria. One of the first identified was the ATP-dependent caseinolytic protease (Clp), whose structure and function have been well characterized in Escherichia coli . Plastid Clp proteins in higher plants are surprisingly numerous and diverse, with at least 16 distinct Clp proteins in the model plant Arabidopsis thaliana . Multiple paralogues exist for several of the different types of plastid Clp protein, with the most extreme being five for the proteolytic subunit ClpP. Both biochemical and genetic studies have recently begun to reveal the intricate structural interactions between the various Clp proteins, and their importance for chloroplast function and plant development. Much of the recent data suggests that the function of many of the Clp proteins probably affects more specific processes within chloroplasts, in addition to the more general 'housekeeping' role previously assumed.