m-AAA protease-driven membrane dislocation allows intramembrane cleavage by rhomboid in mitochondria

Maturation of cytochrome c peroxidase (Ccp1) in mitochondria occurs by the subsequent action of two conserved proteases in the inner membrane: the m-AAA protease, an ATP-dependent protease degrading misfolded proteins and mediating protein processing, and the rhomboid protease Pcp1, an intramembrane cleaving peptidase. Neither the determinants preventing complete proteolysis of certain substrates by the m-AAA protease, nor the obligatory requirement of the m-AAA protease for rhomboid cleavage is currently understood. Here, we describe an intimate and unexpected functional interplay of both proteases. The m-AAA protease mediates the ATP-dependent membrane dislocation of Ccp1 independent of its proteolytic activity. It thereby ensures the correct positioning of Ccp1 within the membrane bilayer allowing intramembrane cleavage by rhomboid. Decreasing the hydrophobicity of the Ccp1 transmembrane segment facilitates its dislocation from the membrane and renders rhomboid cleavage m-AAA protease-independent. These findings reveal for the first time a non-proteolytic function of the m-AAA protease during mitochondrial biogenesis and rationalise the requirement of a preceding step for intramembrane cleavage by rhomboid.     

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.     

Prohibitins regulate membrane protein degradation by the m-AAA protease in mitochondria

Prohibitins comprise a protein family in eukaryotic cells with potential roles in senescence and tumor suppression. Phb1p and Phb2p, members of the prohibitin family in Saccharomyces cerevisiae, have been implicated in the regulation of the replicative life span of the cells and in the maintenance of mitochondrial morphology. The functional activities of these proteins, however, have not been elucidated. We demonstrate here that prohibitins regulate the turnover of membrane proteins by the m-AAA protease, a conserved ATP-dependent protease in the inner membrane of mitochondria. The m-AAA protease is composed of the homologous subunits Yta10p (Afg3p) and Yta12p (Rca1p). Deletion of PHB1 or PHB2 impairs growth of Deltayta10 or Deltayta12 cells but does not affect cell growth in the presence of the m-AAA protease. A prohibitin complex with a native molecular mass of approximately 2 MDa containing Phb1p and Phb2p forms a supercomplex with the m-AAA protease. Proteolysis of nonassembled inner membrane proteins by the m-AAA protease is accelerated in mitochondria lacking Phb1p or Phb2p, indicating a negative regulatory effect of prohibitins on m-AAA protease activity. These results functionally link members of two conserved protein families in eukaryotes to the degradation of membrane proteins in mitochondria.     

Translating m-AAA protease function in mitochondria to hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurodegenerative disorder that is characterized by progressive and cell-specific axonal degeneration. An autosomal recessive form of the disease is caused by mutations in paraplegin, which is a conserved subunit of the ubiquitous and ATP-dependent m-AAA protease in mitochondria. The m-AAA protease carries out protein quality control in the inner membrane of the mitochondria, suggesting a pathogenic role of misfolded proteins in HSP. A recent study demonstrates that the m-AAA protease regulates ribosome assembly and translation within mitochondria by controlling proteolytic maturation of a ribosomal subunit. Here, we will discuss implications of the dual role of the m-AAA protease in protein activation and degradation for mitochondrial dysfunction and axonal degeneration.     

The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria

AAA proteases comprise a conserved family of membrane bound ATP-dependent proteases that ensures the quality control of mitochondrial inner-membrane proteins. Inactivation of AAA proteases causes pleiotropic phenotypes in various organisms, including respiratory deficiencies, mitochondrial morphology defects, and axonal degeneration in hereditary spastic paraplegia (HSP). The molecular basis of these defects, however, remained unclear. Here, we describe a regulatory role of an AAA protease for mitochondrial protein synthesis in yeast. The mitochondrial ribosomal protein MrpL32 is processed by the m-AAA protease, allowing its association with preassembled ribosomal particles and completion of ribosome assembly in close proximity to the inner membrane. Maturation of MrpL32 and mitochondrial protein synthesis are also impaired in a HSP mouse model lacking the m-AAA protease subunit paraplegin, demonstrating functional conservation. Our findings therefore rationalize mitochondrial defects associated with m-AAA protease mutants in yeast and shed new light on the mechanism of axonal degeneration in HSP.     

Presequence-dependent folding ensures MrpL32 processing by the m-AAA protease in mitochondria

m-AAA proteases exert dual functions in the mitochondrial inner membrane: they mediate the processing of specific regulatory proteins and ensure protein quality control degrading misfolded polypeptides to peptides. Loss of these activities leads to neuronal cell death in several neurodegenerative disorders. However, it is unclear how the m-AAA protease chooses between specific processing and complete degradation. A central and conserved function of the m-AAA protease is the processing of the ribosomal subunit MrpL32, which regulates ribosome biogenesis and the formation of respiratory complexes. Here, we demonstrate that the formation of a tightly folded domain harbouring a conserved CxxC-X(9)-CxxC sequence motif halts degradation initiated from the N-terminus and triggers the release of mature MrpL32. Oxidative stress impairs folding of MrpL32, resulting in its degradation by the m-AAA protease and decreased mitochondrial translation. Surprisingly, MrpL32 folding depends on its mitochondrial targeting sequence. Presequence-assisted folding of MrpL32 requires the complete import of the MrpL32 precursor before maturation occurs and therefore explains the need for post-translocational processing by the m-AAA protease rather than co-translocational cleavage by the general mitochondrial processing peptidase.     

Charge Composition Features of Model Single-span Membrane Proteins That Determine Selection of YidC and SecYEG Translocase Pathways in Escherichia coli

We have investigated the features of single-span model membrane proteins based upon leader peptidase that determines whether the proteins insert by a YidC/Sec-independent, YidC-only, or YidC/Sec mechanism. We find that a protein with a highly hydrophobic transmembrane segment that inserts into the membrane by a YidC/Sec-independent mechanism becomes YidC-dependent if negatively charged residues are inserted into the translocated periplasmic domain or if the hydrophobicity of the transmembrane segment is reduced by substituting polar residues for nonpolar ones. This suggests that charged residues in the translocated domain and the hydrophobicity within the transmembrane segment are important determinants of the insertion pathway. Strikingly, the addition of a positively charged residue to either the translocated region or the transmembrane region can switch the insertion requirements such that insertion requires both YidC and SecYEG. To test conclusions from the model protein studies, we confirmed that a positively charged residue is a SecYEG determinant for the endogenous proteins ATP synthase subunits a and b and the TatC subunit of the Tat translocase. These findings provide deeper insights into how pathways are selected for the insertion of proteins into the Escherichia coli inner membrane.     

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.     

Variable and tissue-specific subunit composition of mitochondrial m-AAA protease complexes linked to hereditary spastic paraplegia

The m-AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, controls protein quality and regulates ribosome assembly, thus exerting essential housekeeping functions within mitochondria. Mutations in the m-AAA protease subunit paraplegin cause axonal degeneration in hereditary spastic paraplegia (HSP), but the basis for the unexpected tissue specificity is not understood. Paraplegin assembles with homologous Afg3l2 subunits into hetero-oligomeric complexes which can substitute for yeast m-AAA proteases, demonstrating functional conservation. The function of a third paralogue, Afg3l1 expressed in mouse, is unknown. Here, we analyze the assembly of paraplegin into m-AAA complexes and monitor consequences of paraplegin deficiency in HSP fibroblasts and in a mouse model for HSP. Our findings reveal variability in the assembly of m-AAA proteases in mitochondria in different tissues. Homo-oligomeric Afg3l1 and Afg3l2 complexes and hetero-oligomeric assemblies of both proteins with paraplegin can be formed. Yeast complementation studies demonstrate the proteolytic activity of these assemblies. Paraplegin deficiency in HSP does not result in the loss of m-AAA protease activity in brain mitochondria. Rather, homo-oligomeric Afg3l2 complexes accumulate, and these complexes can substitute for housekeeping functions of paraplegin-containing m-AAA complexes. We therefore propose that the formation of m-AAA proteases with altered substrate specificities leads to axonal degeneration in HSP.     

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.     

SecA-dependence of the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein is a function of sequence context

We have analysed the translocation of a large periplasmic loop in the Escherichia coli MalF Inner membrane protein when placed in different sequence contexts and under conditions when the function of the SecA protein is Inhibited. The results show that the degree of SecA-dependence varies with sequence context: while translocation of the large loop In its normal context Is only minimally affected by SecA Inhibition, translocation is much more sensitive to SecA inhibition when the loop is placed in the context of other inner membrane proteins. Conversely, when the large MalF loop is replaced by segments from other proteins, translocation of those segments is again very sensitive to SecA inhibition. Thus, SecA-dependence is not an all-or-none phenomenon and Is not only a simple function of, e.g. the length of a translocated segment or the hydrophobicity of the flanking transmembrane segments.     

Orientational preferences of neighboring helices can drive ER insertion of a marginally hydrophobic transmembrane helix

α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane α helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (N(cyt)-C(lum) or N(lum)-C(cyt)) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the "missing hydrophobicity" in polytopic membrane proteins.     

Integration of deletion mutants of bovine rhodopsin into the membrane of the endoplasmic reticulum

Newly synthesized eukaryotic membrane proteins must be integrated into the membrane of the endoplasmic reticulum with the correct topology to enable the subsequent acquisition of the correctly folded, functional conformation. Here, an analysis is presented of N-terminal glycosylation and steady-state membrane orientation of a series of truncation mutants of the sevenhelix protein rhodopsin expressed in COS-1 cells. Mutants containing one, three, or five N-terminal transmembrane segments of rhodopsin, as well as mutants containing only the first transmembrane segment, but with hydrophilic extensions at the C-terminus were studied. The findings demonstrate that the C-terminal transmembrane segments play a crucial role in determining the final orientation of rhodopsin, and that the commitment to the correct orientation occurs only after the synthesis of at least three transmembrane segments. The experiments also suggest that the molecular machinery involved in the integration of a newly synthesized seven-helix membrane protein into the endoplasmic reticulum membrane is sensitive to the overall hydrophobicity of the sequence that follows the first transmembrane segment.     

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.     

The phosphate carrier has an ability to be sorted to either the TIM22 pathway or the TIM23 pathway for its import into yeast mitochondria

Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria via the TOM40 (translocase of the mitochondrial outer membrane 40) complex, and follow several distinct sorting pathways to reach their destination submitochondrial compartments. Phosphate carrier (PiC) is an inner membrane protein with 6 transmembrane segments (TM1-TM6) and requires, after translocation across the outer membrane, the Tim9-Tim10 complex and the TIM22 complex to be inserted into the inner membrane. Here we analyzed an in vitro import of fusion proteins between various PiC segments and mouse dihydrofolate reductase. The fusion protein without TM1 and TM2 was translocated across the outer membrane but was not inserted into the inner membrane. The fusion proteins without TM1-TM4 were not inserted into the inner membrane but instead translocated across the inner membrane. Functional defects of Tim50 of the TIM23 complex caused either by depletion of the protein or the addition of anti-Tim50 antibodies blocked translocation of the fusion proteins without TM1-TM4 across the inner membrane, suggesting that lack of TM1-TM4 led to switch of its sorting pathway from the TIM22 pathway to the TIM23 pathway. PiC thus appears to have a latent signal for sorting to the TIM23 pathway, which is exposed by reduced interactions with the Tim9-Tim10 complex and maintenance of the import competence.     

Charged flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p

Mgm1p is a nuclearly encoded GTPase important for mitochondrial fusion. Long and short isoforms of the protein are generated in a unique "alternative topogenesis" process in which the most N-terminal of two hydrophobic segments in the protein is inserted into the inner mitochondrial membrane in about half of the molecules and translocated across the inner membrane in the other half. In the latter population, the second hydrophobic segment is cleaved by the inner membrane protease Pcp1p, generating the short isoform. Here, we show that charged residues in the regions flanking the first segment critically affect the ratio between the two isoforms, providing new insight into the importance of charged residues in the insertion of proteins into the mitochondrial inner membrane.     

TIM23-mediated insertion of transmembrane α-helices into the mitochondrial inner membrane

While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) α-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.     

A Highlights from MBoC Selection: The Hydrophobic Core of the Sec61 Translocon Defines the Hydrophobicity Threshold for Membrane Integration

The Sec61 translocon mediates the translocation of proteins across the endoplasmic reticulum membrane and the lateral integration of transmembrane segments into the lipid bilayer. The structure of the idle translocon is closed by a lumenal plug domain and a hydrophobic constriction ring. To test the function of the apolar constriction, we have mutated all six ring residues of yeast Sec61p to more hydrophilic, bulky, or even charged amino acids (alanines, glycines, serines, tryptophans, lysines, or aspartates). The translocon was found to be surprisingly tolerant even to the charge mutations in the constriction ring, because growth and translocation efficiency were not drastically affected. Most interestingly, ring mutants were found to affect the integration of hydrophobic sequences into the lipid bilayer, indicating that the translocon does not simply catalyze the partitioning of potential transmembrane segments between an aqueous environment and the lipid bilayer but that it also plays an active role in setting the hydrophobicity threshold for membrane integration.     

A higher plant mitochondrial homologue of the yeast m-AAA protease. Molecular cloning,localization, and putative function

Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function. They have been identified in yeast and animals but not yet in plants. This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease. The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH). We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space. Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast. Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.     

Regulation of mitochondrial morphology through proteolytic cleavage of OPA1

The dynamin-like GTPase OPA1, a causal gene product of human dominant optic atrophy, functions in mitochondrial fusion and inner membrane remodeling. It has several splice variants and even a single variant is found as several processed forms, although their functional significance is unknown. In yeast, mitochondrial rhomboid protease regulates mitochondrial function and morphology through proteolytic cleavage of Mgm1, the yeast homolog of OPA1. We demonstrate that OPA1 variants are synthesized with a bipartite-type mitochondrial targeting sequence. During import, the matrix-targeting signal is removed and processed forms (L-isoforms) are anchored to the inner membrane in type I topology. L-isoforms undergo further processing in the matrix to produce S-isoforms. Knockdown of OPA1 induced mitochondrial fragmentation, whose network morphology was recovered by expression of L-isoform but not S-isoform, indicating that only L-isoform is fusion-competent. Dissipation of membrane potential, expression of m-AAA protease paraplegin, or induction of apoptosis stimulated this processing along with the mitochondrial fragmentation. Thus, mammalian mitochondrial function and morphology is regulated through processing of OPA1 in a DeltaPsi-dependent manner.