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.     

The lack of mitochondrial AtFtsH4 protease alters Arabidopsis leaf morphology at the late stage of rosette development under short-day photoperiod

AtFtsH4 is one of four inner membrane-bound mitochondrial ATP-dependent metalloproteases in Arabidopsis thaliana , called AAA proteases, whose catalytic site is exposed to the intermembrane space. In the present study, we used a reverse-genetic approach to investigate the physiological role of the AtFtsH4 protease. We found that loss of AtFtsH4 did not significantly affect Arabidopsis growth under optimal conditions (long days); however, severe morphological and developmental abnormalities in late rosette development occurred under short-day conditions. The asymmetric shape and irregular serration of expanding leaf blades were the most striking features of the ftsh4 mutant phenotype. The severe abnormal morphology of the leaf blades was accompanied by ultrastructural changes in mitochondria and chloroplasts. These abnormalities correlated with elevated levels of reactive oxygen species and carbonylated mitochondrial proteins. We found that two classes of molecular chaperones, Hsp70 and prohibitins, were over-expressed in ftsh4 mutants during late vegetative growth under both short- and long-day conditions. Taken together, our data indicate that lack of AtFtsH4 results in impairment of organelle development and Arabidopsis leaf morphology under short-day conditions.     

The significance of Arabidopsis AAA proteases for activity and assembly/stability of mitochondrial OXPHOS complexes

The Arabidopsis genome encodes four mitochondrially localized adenosine 5'-triphosphate-dependent metalloproteases called FtsH or AAA proteases. All of them span the inner mitochondrial membrane but the catalytic site of two of them (AtFtsH4 and AtFtsH11) faces the intermembrane space, while AtFtsH3 and AtFtsH10 face the matrix. We used a combination of blue-native polyacrylamide gel electrophoresis and histochemical staining to reveal the consequences of the loss of one of mitochondrial FtsHs on the efficiency of the oxidative phosphorylation system in Arabidopsis mitochondria. To address this issue, we have selected homozygous lines of respective transferred DNA (T-DNA) insertional mutants. A decrease in the activity of complexes I and V but not complex IV was observed in the ftsh mutants, except for the mutant lacking functional FtsH11. The reduced activity of complexes I and V was well correlated with a decreased protein level of these complexes. Western blots experiments using specific antibodies against complex V subunits showed a significant reduction of these subunits only in the ftsh4 mutant. Taken together, our results reveal a role of FtsH3, FtsH4 and FtsH10 proteases in the biogenesis of a plant oxidative phosphorylation system.     

ATP-dependent proteases in plant mitochondria: What do we know about them today?

Lon-, Clp- and FtsH-like proteases, members of three families of ATP-dependent proteases derived from bacterial ancestors, have been identified in plant mitochondria. Classifications of mitochondrial-specific paralogues of plant ATP-dependent proteases, based on targeting prediction programs and different experimental methods, are compared. Accumulating evidence points to similarities in the structure and the mechanisms of action used by various ATP-dependent proteases. Therefore, before focusing on plant mitochondrial ATP-dependent proteases, the paper discusses general features of ATP-dependent proteases. To date, information about structure and function of plant mitochondrial Lon-like, Clp-like and FtsH-like proteases is rather scarce, but indicates that these enzymes, like their bacterial and eukaryotic homologues, combine proteolytic and chaperone-like activities to form mitochondrial protein quantity and quality control system in plants.     

Multiple intracellular locations of Lon protease in Arabidopsis: evidence for the localization of AtLon4 to chloroplasts

Arabidopsis contains four Lon protease-like proteins (AtLon1-AtLon4), predicted to be localized in different cellular organelles, including mitochondria, peroxisomes and plastids. A notable question is whether Lon is present in chloroplasts, since it is absent from cyanobacteria and thus appears to have been lost during the evolution of photosynthetic organisms. Based on in vivo transient assays, we found that AtLon4 is dually targeted to both mitochondria and chloroplasts. Furthermore, immunoblot analysis localized AtLon4 to the thylakoids. Thus, in spite of its absence from basal photosynthetic organisms, our results suggest the presence of Lon in plant plastids.     

Assembly factors and ATP-dependent proteases in cytochrome c oxidase biogenesis

Eukaryotic cytochrome c oxidase (CcO), the terminal enzyme of the energy-transducing mitochondrial electron transport chain is a hetero-oligomeric, heme–copper oxidase complex composed of both mitochondrially and nuclear-encoded subunits. It is embedded in the inner mitochondrial membrane where it couples the transfer of electrons from reduced cytochrome c to molecular oxygen with vectorial proton translocation across the membrane. The biogenesis of CcO is a complicated sequential process that requires numerous specific accessory proteins, so-called assembly factors, which include translational activators, translocases, molecular chaperones, copper metallochaperones and heme a biosynthetic enzymes. Besides these CcO-specific protein factors, the correct biogenesis of CcO requires an even greater number of proteins with much broader substrate specificities. Indeed, growing evidence indicates that mitochondrial ATP-dependent proteases might play an important role in CcO biogenesis. Out of the four identified energy-dependent mitochondrial proteases, three were shown to be directly involved in proteolysis of CcO subunits. In addition to their well-established protein-quality control function these oligomeric proteolytic complexes with chaperone-like activities may function as molecular chaperones promoting productive folding and assembly of subunit proteins. In this review, we summarize the current knowledge of the functional involvement of eukaryotic CcO-specific assembly factors and highlight the possible significance for CcO biogenesis of mitochondrial ATP-dependent proteases.     

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.     

AtFtsH heterocomplex-mediated degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to stresses

Chloroplastic heterocomplex consisting of AtFtsH1, 2, 5 and 8 proteases, integrally bound to thylakoid membrane was shown to play a critical role in degradation of photodamaged PsbA molecules, inherent to photosystem II (PSII) repair cycle and in plastid development. As no one thylakoid bound apoproteins besides PsbA has been identified as target for the heterocomplex-mediated degradation we investigated the significance of this protease complex in degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to various stressing conditions and in stress-related changes in overall composition of LHCII trimers of PSII-enriched membranes (BBY particles). To reach this goal a combination of approaches was applied based on immunoblotting, in vitro degradation and non-denaturing isoelectrofocusing. Exposure of Arabidopsis thaliana leaves to desiccation, cold and high irradiance led to a step-wise disappearance of Lhcb1 and Lhcb2, while Lhcb3 level remained unchanged, except for high irradiance which caused significant Lhcb3 decrease. Furthermore, it was demonstrated that stress-dependent disappearance of Lhcb1–3 is a proteolytic phenomenon for which a metalloprotease is responsible. No changes in Lhcb1–3 level were observed due to exposition of var1-1 mutant leaves to the three stresses clearly pointing to the involvement of AtFtsH heterocomplex in the desiccation, cold and high irradiance-dependent degradation of Lhcb1 and Lhcb2 and in high irradiance-dependent degradation of Lhcb3. Non-denaturing isoelectrofocusing analyses revealed that AtFtsH heterocomplex-dependent differential Lhcb1–3 disappearance behaviour following desiccation stress was accompanied by modulations in abundances of individual LHCII trimers of BBY particles and that LHCII of var1-1 resisted the modulations.     

Functional redundancy of AtFtsH metalloproteases in thylakoid membrane complexes

FtsH is an ATP-dependent metalloprotease found in bacteria, mitochondria, and plastids. Arabidopsis (Arabidopsis thaliana) contains 12 AtFtsH proteins, three in the mitochondrion and nine in the chloroplast. Four of the chloroplast FtsH proteins are encoded by paired members of closely related genes (AtFtsH1 and 5, and AtFtsH2 and 8). We have previously reported that AtFtsH2 and 8 are interchangeable components of AtFtsH complexes in the thylakoid membrane. In this article, we show that the var1 variegation mutant, which is defective in AtFtsH5, has a coordinate reduction in the AtFtsH2 and 8 pair, and that the levels of both pairs are restored to normal in var1 plants that overexpress AtFtsH1. Overexpression of AtFtsH1, but not AtFtsH2/VAR2, normalizes the pattern of var1 variegation, restoring a nonvariegated phenotype. We conclude that AtFtsH proteins within a pair, but not between pairs, are interchangeable and functionally redundant, at least in part. We further propose that the abundance of each pair is matched with that of the other pair, with excess subunits being turned over. The variegation phenotype of var1 (as well as var2, which is defective in AtFtsH2) suggests that a threshold concentration of subunits is required for normal chloroplast function. AtFtsH1, 2, 5, and 8 do not show evidence of tissue or developmental specific expression. Phylogenetic analyses revealed that rice (Oryza sativa) and Arabidopsis share a conserved core of seven FtsH subunit genes, including the AtFtsH1 and 5 and AtFtsH2 and 8 pairs, and that the structure of the present-day gene families can be explained by duplication events in each species following the monocot/dicot divergence.     

Protein degradation in mitochondria

The biogenesis of mitochondria and the maintenance of mitochondrial functions depends on an autonomous proteolytic system in the organelle which is highly conserved throughout evolution. Components of this system include processing peptidases and ATP-dependent proteases, as well as molecular chaperone proteins and protein complexes with apparently regulatory functions. While processing peptidases mediate maturation of nuclear-encoded mitochondrial preproteins, quality control within various subcompartments of mitochondria is ensured by ATP-dependent proteases which selectively remove non-assembled or misfolded polypeptides. Moreover; these proteases appear to control the activity- or steady-state levels of specific regulatory proteins and thereby ensure mitochondrial genome integrity, gene expression and protein assembly.     

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.     

Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits.     

Targeting capacity and conservation of PreP homologues localization in mitochondria of different species

Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.     

OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.     

FTSH4 and OMA1 mitochondrial proteases reduce moderate heat stress-induced protein aggregation

The threat of global warming makes uncovering mechanisms of plant tolerance to long-term moderate heat stress particularly important. We previously reported that Arabidopsis (Arabidopsis thaliana) plants lacking mitochondrial proteases FTSH4 or OMA1 suffer phenotypic changes under long-term stress of 30°C, while their growth at 22°C is not affected. Here we found that these morphological and developmental changes are associated with increased accumulation of insoluble mitochondrial protein aggregates that consist mainly of small heat-shock proteins (sHSPs). Greater accumulation of sHSPs in ftsh4 than oma1 corresponds with more severe phenotypic abnormalities. We showed that the proteolytic activity of FTSH4, and to a lesser extent of OMA1, as well as the chaperone function of FTSH4, is crucial for protecting mitochondrial proteins against aggregation. We demonstrated that HSP23.6 and NADH dehydrogenase subunit 9 present in aggregates are proteolytic substrates of FTSH4, and this form of HSP23.6 is also a substrate of OMA1 protease. In addition, we found that the activity of FTSH4 plays an important role during recovery from elevated to optimal temperatures. Isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analyses, along with identification of aggregation-prone proteins, implicated mitochondrial pathways affected by protein aggregation (e.g. assembly of complex I) and revealed that the mitochondrial proteomes of ftsh4 and oma1 plants are similarly adapted to long-term moderate heat stress. Overall, our data indicate that both FTSH4 and OMA1 increase the tolerance of plants to long-term moderate heat stress by reducing detergent-tolerant mitochondrial protein aggregation.

Mitochondrial proteases prevent accumulation of insoluble protein aggregates and protect Arabidopsis plants against long-term moderate heat stress.     

AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria.

The mechanism of selective protein degradation of membrane proteins in mitochondria has been studied employing a model protein that is subject to rapid proteolysis within the inner membrane. Protein degradation was mediated by two different proteases: (i) the m-AAA protease, a protease complex consisting of multiple copies of the ATP-dependent metallopeptidases Yta1Op (Afg3p) and Yta12p (Rcalp); and (ii) by Ymelp (Ytallp) that also is embedded in the inner membrane. Ymelp, highly homologous to Yta1Op and Yta12p, forms a complex of approximately 850 kDa in the inner membrane and exerts ATP-dependent metallopeptidase activity. While the m-AAA protease exposes catalytic sites to the mitochondrial matrix, Ymelp is active in the intermembrane space. The Ymelp complex was therefore termed 'i-AAA protease'. Analysis of the proteolytic fragments indicated cleavage of the model polypeptide at the inner and outer membrane surface and within the membrane-spanning domain. Thus, two AAA proteases with their catalytic sites on opposite membrane surfaces constitute a novel proteolytic system for the degradation of membrane proteins in mitochondria.