Substrate recognition by AAA+ ATPases: distinct substrate binding modes in ATP-dependent protease Yme1 of the mitochondrial intermembrane space

The energy-dependent proteolysis of cellular proteins is mediated by conserved proteolytic AAA(+) complexes. Two such machines, the m- and i-AAA proteases, are present in the mitochondrial inner membrane. They exert chaperone-like properties and specifically degrade nonnative membrane proteins. However, molecular mechanisms of substrate engagement by AAA proteases remained elusive. Here, we define initial steps of substrate recognition and identify two distinct substrate binding sites in the i-AAA protease subunit Yme1. Misfolded polypeptides are recognized by conserved helices in proteolytic and AAA domains. Structural modeling reveals a lattice-like arrangement of these helices at the surface of hexameric AAA protease ring complexes. While helices within the AAA domain apparently play a general role for substrate binding, the requirement for binding to surface-exposed helices within the proteolytic domain is determined by the folding and membrane association of substrates. Moreover, an assembly factor of cytochrome c oxidase, Cox20, serves as a substrate-specific cofactor during proteolysis and modulates the initial interaction of nonassembled Cox2 with the protease. Our findings therefore reveal the existence of alternative substrate recognition pathways within AAA proteases and shed new light on molecular mechanisms ensuring the specificity of proteolysis by energy-dependent proteases.     

Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface

Two AAA proteases, each with its catalytic site at the opposite membrane surface, mediate the ATP-dependent degradation of mitochondrial inner membrane proteins. We demonstrate here that a model substrate polypeptide containing hydrophilic domains at both sides of the membrane can be completely degraded by either of the AAA proteases, if solvent-exposed domains are in an unfolded state. A short protein tail protruding from the membrane surface is sufficient to allow the proteolytic attack of an AAA protease that facilitates domain unfolding at the opposite side. Our results provide a rationale for the membrane arrangement of AAA proteases in mitochondria and demonstrate that degradation of membrane proteins by AAA proteases involves an active extraction of transmembrane segments and transport of solvent-exposed domains across the membrane.     

MAP-1 and IAP-1, two novel AAA proteases with catalytic sites on opposite membrane surfaces in mitochondrial inner membrane of Neurospora crassa

Eukaryotic AAA proteases form a conserved family of membrane-embedded ATP-dependent proteases but have been analyzed functionally only in the yeast Saccharomyces cerevisiae. Here, we have identified two novel members of this protein family in the filamentous fungus Neurospora crassa, which were termed MAP-1 and IAP-1. Both proteins are localized to the inner membrane of mitochondria. They are part of two similar-sized high molecular mass complexes, but expose their catalytic sites to opposite membrane surfaces, namely, the intermembrane and the matrix space. Disruption of iap-1 by repeat-induced point mutation caused a slow growth phenotype at high temperature and stabilization of a misfolded inner membrane protein against degradation. IAP-1 could partially substitute for functions of its yeast homolog Yme1, demonstrating functional conservation. However, respiratory growth at 37 degrees C was not restored. Our results identify two components of the quality control system of the mitochondrial inner membrane in N. crassa and suggest that AAA proteases with catalytic sites exposed to opposite membrane surfaces are present in mitochondria of all eukaryotic cells.     

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.     

Oma1, a novel membrane-bound metallopeptidase in mitochondria with activities overlapping with the m-AAA protease

The integrity of the inner membrane of mitochondria is maintained by a membrane-embedded quality control system that ensures the removal of misfolded membrane proteins. Two ATP-dependent AAA proteases with catalytic sites at opposite membrane surfaces are key components of this proteolytic system. Here we describe the identification of a novel conserved metallopeptidase that exerts activities overlapping with the m-AAA protease and was therefore termed Oma1. Both peptidases are integral parts of the inner membrane and mediate the proteolytic breakdown of a misfolded derivative of the polytopic inner membrane protein Oxa1. The m-AAA protease cleaves off the matrix-exposed C-terminal domain of Oxa1 and processively degrades its transmembrane domain. In the absence of the m-AAA protease, proteolysis of Oxa1 is mediated in an ATP-independent manner by Oma1 and a yet unknown peptidase resulting in the accumulation of N- and C-terminal proteolytic fragments. Oma1 exposes its proteolytic center to the matrix side; however, mapping of Oma1 cleavage sites reveals clipping of Oxa1 in loop regions at both membrane surfaces. These results identify Oma1 as a novel component of the quality control system in the inner membrane of mitochondria. Proteins homologous to Oma1 are present in higher eukaryotic cells, eubacteria and archaebacteria, suggesting that Oma1 is the founding member of a conserved family of membrane-embedded metallopeptidases.     

Membrane protein turnover by the m-AAA protease in mitochondria depends on the transmembrane domains of its subunits

AAA proteases are membrane-bound ATP-dependent proteases that are present in eubacteria, mitochondria and chloroplasts and that can degrade membrane proteins. Recent evidence suggests dislocation of membrane-embedded substrates for proteolysis to occur in a hydrophilic environment; however, next to nothing is known about the mechanism of this process. Here, we have analysed the role of the membrane-spanning domains of Yta10 and Yta12, which are conserved subunits of the hetero-oligomeric m-AAA protease in the mitochondria of Saccharomyces cerevisiae. We demonstrate that the m-AAA protease retains proteolytic activity after deletion of the transmembrane segments of either Yta10 or Yta12. Although the mutant m-AAA protease is still capable of processing cytochrome c peroxidase and degrading a peripheral membrane protein, proteolysis of integral membrane proteins is impaired. We therefore propose that transmembrane segments of m-AAA protease subunits have a direct role in the dislocation of membrane-embedded substrates.     

Localization of general and regulatory proteolysis in Bacillus subtilis cells

Protein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation.     

Identification and characterization of YME1L1, a novel paraplegin-related gene

A gene responsible for an autosomal recessive form of hereditary spastic paraplegia (SPG7) was recently identified. This gene encodes paraplegin, a mitochondrial protein highly homologous to the yeast mitochondrial AAA proteases Afg3p, Rca1p, and Yme1p, which have both proteolytic and chaperone-like activities at the inner mitochondrial membrane. By screening the expressed sequence tag database, we identified and characterized a novel human gene, YME1L1 (YME1L1-like1, HGMW-approved symbol). This gene encodes a predicted protein of 716 amino acids highly similar to all mitochondrial AAA proteases and in particular to yeast Yme1p. Expression and immunofluorescence studies revealed that YME1L1 and paraplegin share a similar expression pattern and the same subcellular localization in the mitochondrial compartment. YME1L1 may represent a candidate gene for other forms of hereditary spastic paraplegia and possibly for other neurodegenerative disorders.     

Membrane protein degradation by AAA proteases in mitochondria

The inner membrane of mitochondria is one of the protein's richest cellular membranes. The biogenesis of the respiratory chain and ATP-synthase complexes present in this membrane is an intricate process requiring the coordinated function of various membrane-bound proteins including protein translocases and assembly factors. It is therefore not surprising that a distinct quality control system is present in this membrane that selectively removes nonassembled polypeptides and prevents their possibly deleterious accumulation in the membrane. The key components of this system are two AAA proteases, membrane-embedded ATP-dependent proteolytic complexes, which expose their catalytic sites at opposite membrane surfaces. Other components include the prohibitin complex with apparently chaperone-like properties and a regulatory function during proteolysis and a recently identified ATP-binding cassette (ABC) transporter that exports peptides derived from the degradation of membrane proteins from the matrix to the intermembrane space. All of these components are highly conserved during evolution and appear to be ubiquitously present in mitochondria of eukaryotic cells, indicating important cellular functions. This review will summarize our current understanding of this proteolytic system and, in particular, focus on the mechanisms guiding the degradation of membrane proteins by AAA proteases.     

AAA proteases: cellular machines for degrading membrane proteins

AAA proteases are a conserved class of ATP-dependent proteases that mediate the degradation of membrane proteins in bacteria, mitochondria and chloroplasts. They combine proteolytic and chaperone-like activities and thus form a membrane-integrated quality-control system. Inactivation of AAA proteases causes severe defects in various organisms, including neurodegeneration in humans. Proteolysis by AAA proteases is modulated by another membrane-protein complex that is composed of prohibitins in eukaryotic cells and related proteins in bacteria.     

Characterization of mutants of the Escherichia coli AAA protease, FtsH, carrying a mutation in the central pore region

Escherichia coli FtsH is an ATP-dependent and membrane-bound protease, which belongs to the ATPases associated with diverse cellular activities family. FtsH degrades a subset of cytoplasmic regulatory proteins and misassembled membrane proteins. It has been proposed that ATP-dependent proteases unfold and translocate substrate proteins into the protease chamber. Previously, we reported that Phe228 and Gly230 in the conserved motif, @XG (where @ is an aromatic residue and X is any residue), in the central pore of the FtsH ATPase ring have important roles in proteolysis and its coupling to ATP hydrolysis. In this paper, we constructed and characterized additional pore mutants. Results indicated that certain acidic residues located in the pore region are also important for the activity of FtsH. Proteolytic activities of most mutants are correlated with their ATPase activities. Evidence also indicated that Val229, the 2nd residue of the @XG motif, may have a substrate-specific role.     

Flavodoxin, a new fluorescent substrate for monitoring proteolytic activity of FtsH lacking a robust unfolding activity

Escherichia coli FtsH, which belongs to the ATPases associated with diverse cellular activities (AAA) family, is an ATP-dependent and membrane-bound protease. FtsH degrades misassembled membrane proteins and a subset of cytoplasmic regulatory proteins. To elucidate the molecular mechanisms of the proteolysis, a system for precisely monitoring substrate degradation is required. We have exploited E. coli flavodoxin containing non-covalently bound flavin mononucleotide (FMN) as a model substrate for monitoring protein degradation. It was found that FtsH degrades FMN-free apo-flavodoxin but not holo-flavodoxin. However, degradation of a mutant flavodoxin carrying a substitution of Tyr94 to Asp with a lower affinity for FMN could be monitored by fluorimetry. This newly developed monitoring system will also be applicable for proteolysis by other ATP-dependent proteases.     

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.     

Archaeal proteasomes: potential in metabolic engineering

Archaea are a valuable source of enzymes for industrial and scientific applications because of their ability to survive extreme conditions including high salt and temperature. Thanks to advances in molecular biology and genetics, archaea are also attractive hosts for metabolic engineering. Understanding how energy-dependent proteases and chaperones function to maintain protein quality control is key to high-level synthesis of recombinant products. In archaea, proteasomes are central players in energy-dependent proteolysis and form elaborate nanocompartments that degrade proteins into oligopeptides by processive hydrolysis. The catalytic core responsible for this proteolytic activity is the 20S proteasome, a barrel-shaped particle with a central channel and axial gates on each end that limit substrate access to a central proteolytic chamber. AAA proteins (ATPases associated with various cellular activities) are likely to play several roles in mediating energy-dependent proteolysis by the proteasome. These include ATP binding/hydrolysis, substrate binding/unfolding, opening of the axial gates, and translocation of substrate into the proteolytic chamber.     

Molecular Mechanism of Polypeptide Translocation Catalyzed by the Escherichia coli ClpA Protein Translocase

The removal of damaged or unneeded proteins by ATP-dependent proteases is crucial for cell survival in all organisms. Integral components of ATP-dependent proteases are motor proteins that unfold stably folded proteins that have been targeted for removal. These protein unfoldases/polypeptide translocases use ATP to unfold the target proteins and translocate them into a proteolytic component. Despite the central role of these motor proteins in cell homeostasis, a number of important questions regarding the molecular mechanisms of enzyme catalyzed protein unfolding and translocation remain unanswered. Here, we demonstrate that Escherichia coli ClpA, in the absence of the proteolytic component ClpP, processively and directionally steps along the polypeptide backbone with a kinetic step size of ∼ 14 amino acids, independent of the concentration of ATP with a rate of ∼ 19 amino acids s⁻¹ at saturating concentrations of ATP. In contrast to earlier studies by others, we have developed single-turnover fluorescence stopped-flow methods that allow us to quantitatively examine the molecular mechanism of the motor component ClpA decoupled from the proteolytic component ClpP. For the first time, we reveal that in the absence of ClpP ClpA translocates polypeptides directionally, processively and in discrete steps similar to other motor proteins that translocate vectorially on a linear lattice, such as nucleic acid helicases and kinesin. We believe that the methods employed here will be generally applicable to the examination of other AAA?+ protein translocases involved in a variety of important biological functions where the substrate is not covalently modified; for example, membrane fusion, membrane transport, protein disaggregation, and protein refolding.     

Role of the AAA protease Yme1 in folding of proteins in the intermembrane space of mitochondria

The vast majority of mitochondrial proteins are synthesized in the cytosol and transported into the organelle in a largely, if not completely, unfolded state. The proper function of mitochondria thus depends on folding of several hundreds of proteins in the various subcompartments of the organelle. Whereas folding of proteins in the mitochondrial matrix is supported by members of several chaperone families, very little is known about folding of proteins in the intermembrane space (IMS). We targeted dihydrofolate reductase (DHFR) as a model substrate to the IMS of yeast mitochondria and analyzed its folding. DHFR can fold in this compartment, and its aggregation upon heat shock can be prevented in an ATP-dependent manner. Yme1, an AAA (ATPases associated with diverse cellular activities) protease of the IMS, prevented aggregation of DHFR. Analysis of protein aggregates in mitochondria lacking Yme1 revealed the presence of a number of proteins involved in the establishment of mitochondrial ultrastructure, lipid metabolism, protein import, and respiratory growth. These findings explain the pleiotropic effects of deletion of YME1 and suggest an important role for Yme1 as a folding assistant, in addition to its proteolytic function, in the protein homeostasis of mitochondria     

Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines

Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines.     

Proteomic analysis of mitochondrial protein turnover: identification of novel substrate proteins of the matrix protease pim1

ATP-dependent oligomeric proteases are major components of cellular protein quality control systems. To investigate the role of proteolytic processes in the maintenance of mitochondrial functions, we analyzed the dynamic behavior of the mitochondrial proteome of Saccharomyces cerevisiae by two-dimensional (2D) polyacrylamide gel electrophoresis. By a characterization of the influence of temperature on protein turnover in isolated mitochondria, we were able to define four groups of proteins showing a differential susceptibility to proteolysis. The protein Pim1/LON has been shown to be the main protease in the mitochondrial matrix responsible for the removal of damaged or nonnative proteins. To assess the substrate range of Pim1 under in vivo conditions, we performed a quantitative comparison of the 2D protein spot patterns between wild-type and pim1Delta mitochondria. We were able to identify a novel subset of mitochondrial proteins that are putative endogenous substrates of Pim1. Using an in organello degradation assay, we confirmed the Pim1-specific, ATP-dependent proteolysis of the newly identified substrate proteins. We could demonstrate that the functional integrity of the Pim1 substrate proteins, in particular, the presence of intact prosthetic groups, had a major influence on the susceptibility to proteolysis.     

Protein degradation within mitochondria: versatile activities of AAA proteases and other peptidases

Cell survival depends on essential processes in mitochondria. Various proteases within these organelles regulate mitochondrial biogenesis and ensure the complete degradation of excess or damaged proteins. Many of these proteases are highly conserved and ubiquitous in eukaryotic cells. They can be assigned to three functional classes: processing peptidases, which cleave off mitochondrial targeting sequences of nuclearly encoded proteins and process mitochondrial proteins with regulatory functions; ATP-dependent proteases, which either act as processing peptidases with regulatory functions or as quality-control enzymes degrading non-native polypeptides to peptides; and oligopeptidases, which degrade these peptides and mitochondrial targeting sequences to amino acids. Disturbances of protein degradation within mitochondria cause severe phenotypes in various organisms and can lead to the induction of apoptotic programmes and cell-specific neurodegeneration in mammals. After an overview of the proteolytic system of mitochondria, we will focus on versatile functions of ATP-dependent AAA proteases in the inner membrane. These conserved proteolytic machines conduct protein quality surveillance of mitochondrial inner membrane proteins, mediate vectorial protein dislocation from membranes, and, acting as processing enzymes, control ribosome assembly, mitochondrial protein synthesis, and mitochondrial fusion. Implications of these functions for cell-specific axonal degeneration in hereditary spastic paraplegia will be discussed.     

Structural properties of substrate proteins determine their proteolysis by the mitochondrial AAA+ protease Pim1

The protease Pim1/LON, a member of the AAA+ family of homo-oligomeric ATP-dependent proteases, is responsible for the degradation of soluble proteins in the mitochondrial matrix. To establish the molecular parameters required for the specific recognition and proteolysis of substrate proteins by Pim1, we analyzed the in organello degradation of imported reporter proteins containing different structural properties. The amino acid composition at the amino-terminal end had no major effect on the proteolysis reaction. However, proteins with an amino-terminal extension of less than 60 amino acids in front of a stably folded reporter domain were completely resistant to proteolysis by Pim1. Substrate proteins with a longer amino-terminal extension showed incomplete proteolysis, resulting in the generation of a defined degradation fragment. We conclude that Pim1-mediated protein degradation is processive and is initiated from an unstructured amino-terminal segment. Resistance to degradation and fragment formation was abolished if the folding state of the reporter domain was destabilized, indicating that Pim1 is not able to unravel folded proteins for proteolysis. We propose that the requirement for an exposed, large, non-native protein segment, in combination with a limited unfolding capability, accounts for the selectivity of the protease Pim1 for damaged or misfolded polypeptides.