A new function in translocation for the mitochondrial i-AAA protease Yme1: import of polynucleotide phosphorylase into the intermembrane space

Polynucleotide phosphorylase (PNPase) is an exoribonuclease and poly(A) polymerase postulated to function in the cytosol and mitochondrial matrix. Prior overexpression studies resulted in PNPase localization to both the cytosol and mitochondria, concurrent with cytosolic RNA degradation and pleiotropic cellular effects, including growth inhibition and apoptosis, that may not reflect a physiologic role for endogenous PNPase. We therefore conducted a mechanistic study of PNPase biogenesis in the mitochondrion. Interestingly, PNPase is localized to the intermembrane space by a novel import pathway. PNPase has a typical N-terminal targeting sequence that is cleaved by the matrix processing peptidase when PNPase engaged the TIM23 translocon at the inner membrane. The i-AAA protease Yme1 mediated translocation of PNPase into the intermembrane space but did not degrade PNPase. In a yeast strain deleted for Yme1 and expressing PNPase, nonimported PNPase accumulated in the cytosol, confirming an in vivo role for Yme1 in PNPase maturation. PNPase localization to the mitochondrial intermembrane space suggests a unique role distinct from its highly conserved function in RNA processing in chloroplasts and bacteria. Furthermore, Yme1 has a new function in protein translocation, indicating that the intermembrane space harbors diverse pathways for protein translocation.     

Loss of Yme1L perturbates mitochondrial dynamics

Yme1L is an AAA protease that is embedded in the mitochondrial inner membrane with its catalytic domain facing the mitochondrial inner-membrane space. However, how Yme1L regulates mammalian mitochondrial function is still obscure. We find that endogenous Yme1L locates at punctate structures of mitochondria, and that loss of Yme1L in mouse embryonic fibroblast (MEF) cells results in mitochondrial fragmentation and leads to significant increased ‘kiss-and-run'' type of mitochondrial fusion; however, Yme1L knockdown (shYme1L (short hairpin-mediated RNA interference of Yme1L)) cells still remain normal mitochondrial fusion although shYme1L mitochondria have a little bit less fusion and fission rates, and the shYme1L-induced fragmentation is due to a little bit more mitochondrial fission than fusion in cells. Furthermore, shYme1L-induced mitochondrial fragmentation is independent on optic atrophy 1 (OPA1) S1 or S2 processing, and shYme1L results in the stabilization of OPA1 long form (L-OPA1); in addition, the exogenous expression of OPA1 or L-OPA1 facilitates the shYme1L-induced mitochondrial fragmentation, thus this fragmentation induced by shYme1L appears to be associated with L-OPA1''s stability. ShYme1L also causes a slight increase of mitochondrial dynamics proteins of 49 kDa and mitochondrial fission factor (Mff), which recruit mitochondrial key fission factor dynamin-related protein 1 (Drp1) into mitochondria in MEF cells, and loss of Drp1 or Mff inhibits the shYme1L-induced mitochondrial fragmentation. In addition, there is interaction between SLP-2 with Yme1L and shYme1L cells retain stress-induced mitochondrial hyperfusion. Taken together, our results clarify how Yme1L regulates mitochondrial morphology.     

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     

A Tag at the Carboxy Terminus Prevents Membrane Integration of VDAC1 in Mammalian Mitochondria

β-Barrel proteins are found in the outer membranes of bacteria, chloroplasts and mitochondria. The evolutionary conserved sorting and assembly machinery (SAM complex) assembles mitochondrial β-barrel proteins, such as voltage-dependent anion-selective channel 1 (VDAC1), into complexes in the outer membrane by recognizing a sorting β-signal in the carboxy-terminal part of the protein. Here we show that in mammalian mitochondria, masking of the C-terminus of β-barrel proteins by a tag leads to accumulation of soluble misassembled protein in the intermembrane space, which causes mitochondrial fragmentation and loss of membrane potential. A similar phenotype is observed if the β-signal is shortened, removed or when the conserved hydrophobic residues in the β-signal are mutated. The length of the tag at the C-terminus is critical for the assembly of VDAC1, as well as the amino acid residues at positions 130, 222, 225 and 251 of the protein. We propose that if the recognition of the β-signal or the folding of the β-barrel proteins is inhibited, the nonassembled protein will accumulate in the intermembrane space, aggregate and damage mitochondria. This effect offers easy tools for studying the requirements for the membrane assembly of β-barrel proteins, but also advises caution when interpreting the outcome of the β-barrel protein overexpression experiments.     

PARL partitions the lipid transfer protein STARD7 between the cytosol and mitochondria

Intramembrane‐cleaving peptidases of the rhomboid family regulate diverse cellular processes that are critical for development and cell survival. The function of the rhomboid protease PARL in the mitochondrial inner membrane has been linked to mitophagy and apoptosis, but other regulatory functions are likely to exist. Here, we identify the START domain‐containing protein STARD7 as an intramitochondrial lipid transfer protein for phosphatidylcholine. We demonstrate that PARL‐mediated cleavage during mitochondrial import partitions STARD7 to the cytosol and the mitochondrial intermembrane space. Negatively charged amino acids in STARD7 serve as a sorting signal allowing mitochondrial release of mature STARD7 upon cleavage by PARL. On the other hand, membrane insertion of STARD7 mediated by the TIM23 complex promotes mitochondrial localization of mature STARD7. Mitochondrial STARD7 is necessary and sufficient for the accumulation of phosphatidylcholine in the inner membrane and for the maintenance of respiration and cristae morphogenesis. Thus, PARL preserves mitochondrial membrane homeostasis via STARD7 processing and is emerging as a critical regulator of protein localization between mitochondria and the cytosol.     

Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells

Cytochrome c release from mitochondria is central to apoptosis, but the events leading up to it are disputed. The mitochondrial membrane potential has been reported to decrease, increase or remain unchanged during cytochrome c release. We measured mitochondrial membrane potential in Jurkat cells undergoing apoptosis by the uptake of the radiolabelled lipophilic cation TPMP, enabling small changes in potential to be determined. The ATP/ADP ratio, mitochondrial and cell volumes, plasma membrane potential and the mitochondrial membrane potential in permeabilised cells were also measured. Before cytochrome c release the mitochondrial membrane potential increased, followed by a decrease in potential associated with mitochondrial swelling and the release of cytochrome c and DDP-1, an intermembrane space house keeping protein. Mitochondrial swelling and cytochrome c release were both blocked by bongkrekic acid, an inhibitor of the permeability transition. We conclude that during apoptosis mitochondria undergo an initial priming phase associated with hyperpolarisation which leads to an effector phase, during which mitochondria swell and release cytochrome c.     

Regulation of mitochondrial compartment volumes in rat adrenal cortex by ether stress

In vivo ether stress of rats causes release of pituitary adrenocorticotropin (ACTH) leading to activation of steroidogenesis in adrenal cortex mitochondria. The present studies show that this treatment also induces a decrease in the volume of the intermembrane space in isolated adrenal mitochondria. This decrease is accompanied by an increase in the volume of the matrix, thus leaving the total mitochondrial volume approximately constant. These effects are prevented by the protein synthesis inhibitor, cycloheximide, and are specific to the adrenal gland. The decrease in the intermembrane space (or increase in the matrix volume) is correlated with activation of the cholesterol side chain cleavage reaction (the regulated step in steroidogenesis). We propose as a working hypothesis that these changes reflect a hormonally regulated alteration in the relationship between the outer and inner mitochondrial membranes, which may facilitate the rate-limiting movement of cholesterol from the outer to the inner membrane where the side chain cleavage enzyme is located.     

Mitochondrial regulation of cell death: Processing of apoptosis-inducing factor (AIF)

Apoptosis might proceed through the activation of both caspase-dependent and -independent pathways. Apoptosis-inducing factor (AIF) was discovered as the first protein that mediated caspase-independent cell death. Initially, it was regarded as a soluble protein residing in the intermembrane space of mitochondria, from where it could be exported to the nucleus to participate in large-scale DNA fragmentation and chromatin condensation. However, later it was demonstrated that AIF is N-terminally anchored to the inner mitochondrial membrane. Hence, AIF must be liberated from its membrane anchor prior to being released into the cytosol. The current knowledge about the molecular mechanisms regulating the processing and release of AIF from the mitochondria will be summarized and discussed in this review.     

The mitochondrial gateway to cell death

Mitochondria play a key role in death signaling. The intermembrane space of these organelles contains a number of proteins which promote cell death once they are redistributed to the cytosol. The formation of pores in the outer membrane of mitochondria defines a gateway through which the apoptogenic proteins pass during death signaling. Interactions between pro-apoptotic and pro-survival members of the Bcl-2 family of proteins are decisive in the initiation of pore opening. While the specific composition of the pore in molecular terms is still subject to debate and continuing investigation, it is recognized functionally as a passive channel which not only allows egress of proteins to cytosol but also entry in the reverse direction. A variety of constraints may restrict the release of proteins from the intermembrane space to the cytosol. These include trapping in the intercristal spaces formed by the convoluted invaginations of the inner membrane, binding of proteins to the inner membrane or to other soluble proteins of the intermembrane space, or insertion of proteins into the inner membrane. There is a corresponding variety of mechanisms that facilitate release of apoptogenic proteins from such entrapment. Morphological changes that expand the inner membrane enable proteins to be released from enclosure in intercristal spaces, allowing these proteins access to the mitochondrial gateway. Specific cases include cytochrome c molecules bound to inner membrane cardiolipin and released upon oxidation of that lipid component. Further, AIF that is embedded in the inner membrane is released by proteases (caspases or calpains), which enter from the cytosol once the outer membrane pore has opened. The facilitation (or restriction) of apoptogenic protein release through the mitochondrial gateway may provide new opportunities for regulating cell death.     

YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis, and cell proliferation

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.     

Role of the mitochondrial permeability transition and cytochrome C release in hydrogen peroxide-induced apoptosis

We investigated the role of the mitochondrial inner membrane permeability transition and subsequent release of cytochrome c into the cytosol during oxidative stress-evoked apoptosis. Sublethal oxidative stress was applied by treating L929 cells with 0.5 mM H2O2 for 90 min. Then the cellular localization of cytochrome c was examined by immunofluorescent staining and Western blotting. H2O2 treatment caused the permeability transition and pore formation, resulting in membrane depolarization and translocation of cytochrome c from the mitochondria into the cytosol. Pretreatment with cyclosporin A and aristolochic acid (to inhibit pore formation) significantly attenuated a reduction of the mitochondrial membrane potential, as well as signs of apoptosis such as DNA fragmentation, increased plasma membrane permeability, and chromatin condensation. Therefore, exposure to H2O2 caused the opening of permeability transition pores in the inner mitochondrial membrane. An essential role of cytosolic cytochrome c in the execution of apoptosis was demonstrated by its direct microinjection into the cytosol, thus bypassing the need for cytochrome c release from the mitochondrial intermembrane space. Microinjection of cytochrome c caused caspase-dependent apoptosis.     

A cut short to death: Parl and Opa1 in the regulation of mitochondrial morphology and apoptosis

Mitochondria are crucial amplifiers of death signals. They release cytochrome c and other pro-apoptotic factors required to fully activate effector caspases. This release is accompanied by fragmentation of the mitochondrial reticulum and by remodelling of the internal structure of the organelle. Here we review data supporting the existence of a regulatory network in the inner mitochondrial membrane that includes optic atrophy 1 (Opa1), a dynamin-related protein, and presenilin-associated rhomboid-like (Parl), a rhomboid protease. Opa1 regulates remodelling of the cristae independent of its effect on fusion. Cristae remodelling conversely requires Parl, which participates in the production of a soluble form of Opa1 retrieved together with the integral membrane one in oligomers that are disrupted early during apoptosis. Parl itself is regulated by proteolysis to generate a cleaved form, which in turn modulates the shape of the mitochondrial reticulum. Cleavage of Parl depends on its phosphorylation state around the cleavage site, implicating mitochondrial kinases and phosphatases in the regulation of mitochondrial shape.     

Calcium induced release of mitochondrial cytochrome c by different mechanisms selective for brain versus liver.

Increased mitochondrial Ca2+ accumulation is a trigger for the release of cytochrome c from the mitochondrial intermembrane space into the cytosol where it can activate caspases and lead to apoptosis. This study tested the hypothesis that Ca2+-induced release of cytochrome c in vitro can occur by membrane permeability transition (MPT)-dependent and independent mechanisms, depending on the tissue from which mitochondria are isolated. Mitochondria were isolated from rat liver and brain and suspended at 37 degrees C in a K+-based medium containing oxidizable substrates, ATP, and Mg2+. Measurements of changes in mitochondrial volume (via light scattering and electron microscopy), membrane potential and the medium free [Ca2+] indicated that the addition of 0.3 - 3.2 micromol Ca2+ mg-1 protein induced the MPT in liver but not brain mitochondria. Under these conditions, a Ca2+ dose-dependent release of cytochrome c was observed with both types of mitochondria; however, the MPT inhibitor cyclosporin A was only capable of inhibiting this release from liver mitochondria. Therefore, the MPT is responsible for cytochrome c release from liver mitochondria, whereas an MPT-independent mechanism is responsible for release from brain mitochondria.     

Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space

The mitochondrial inner membrane contains numerous multispanning integral proteins. The precursors of these hydrophobic proteins are synthesized in the cytosol and therefore have to cross the mitochondrial outer membrane and intermembrane space to reach the inner membrane. While the import pathways of noncleavable multispanning proteins, such as the metabolite carriers, have been characterized in detail by the generation of translocation intermediates, little is known about the mechanism by which cleavable preproteins of multispanning proteins, such as Oxa1, are transferred from the outer membrane to the inner membrane. We have identified a translocation intermediate of the Oxa1 preprotein in the translocase of the outer membrane (TOM) and found that there are differences from the import mechanisms of carrier proteins. The intermembrane space domain of the receptor Tom22 supports the stabilization of the Oxa1 intermediate. Transfer of the Oxa1 preprotein to the inner membrane is not affected by inactivation of the soluble TIM complexes. Both the inner membrane potential and matrix heat shock protein 70 are essential to release the preprotein from the TOM complex, suggesting a close functional cooperation of the TOM complex and the presequence translocase of the inner membrane. We conclude that mitochondria employ different mechanisms for translocation of multispanning proteins across the aqueous intermembrane space.     

The mitochondrial TOM complex is required for tBid/Bax-induced cytochrome c release

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.     

Down‐regulation of the mitochondrial i‐AAA protease Yme1L induces muscle atrophy via FoxO3a and myostatin activation

Muscle atrophy is closely associated with many diseases, including diabetes and cardiac failure. Growing evidence has shown that mitochondrial dysfunction is related to muscle atrophy; however, the underlying mechanisms are still unclear. To elucidate how mitochondrial dysfunction causes muscle atrophy, we used hindlimb‐immobilized mice. Mitochondrial function is optimized by balancing mitochondrial dynamics, and we observed that this balance shifted towards mitochondrial fission and that MuRF1 and atrogin‐1 expression levels were elevated in these mice. We also found that the expression of yeast mitochondrial escape 1‐like ATPase (Yme1L), a mitochondrial AAA protease was significantly reduced both in hindlimb‐immobilized mice and carbonyl cyanide m‐chlorophenylhydrazone (CCCP)‐treated C2C12 myotubes. When Yme1L was depleted in myotubes, the short form of optic atrophy 1 (Opa1) accumulated, leading to mitochondrial fragmentation. Moreover, a loss of Yme1L, but not of LonP1, activated AMPK and FoxO3a and concomitantly increased MuRF1 in C2C12 myotubes. Intriguingly, the expression of myostatin, a myokine responsible for muscle protein degradation, was significantly increased by the transient knock‐down of Yme1L. Taken together, our results suggest that a deficiency in Yme1L and the consequential imbalance in mitochondrial dynamics result in the activation of FoxO3a and myostatin, which contribute to the pathological state of muscle atrophy.     

Stoichiometric expression of mtHsp40 and mtHsp70 modulates mitochondrial morphology and cristae structure via Opa1L cleavage

Deregulation of mitochondrial heat-shock protein 40 (mtHsp40) and dysfunction of mtHsp70 are associated with mitochondrial fragmentation, suggesting that mtHsp40 and mtHsp70 may play roles in modulating mitochondrial morphology. However, the mechanism of mitochondrial fragmentation induced by mtHsp40 deregulation and mtHsp70 dysfunction remains unclear. In addition, the functional link between mitochondrial morphology change upon deregulated mtHsp40/mtHsp70 and mitochondrial function has been unexplored. Our coimmunoprecipitation and protein aggregation analysis showed that both overexpression and depletion of mtHsp40 accumulated aggregated proteins in fragmented mitochondria. Moreover, mtHsp70 loss and expression of a mtHsp70 mutant lacking the client-binding domain caused mitochondrial fragmentation. Together the data suggest that the molecular ratio of mtHsp40 to mtHsp70 is important for their chaperone function and mitochondrial morphology. Whereas mitochondrial translocation of Drp1 was not altered, optic atrophy 1 (Opa1) short isoform accumulated in fragmented mitochondria, suggesting that mitochondrial fragmentation in this study results from aberration of mitochondrial inner membrane fusion. Finally, we found that fragmented mitochondria were defective in cristae development, OXPHOS, and ATP production. Taken together, our data suggest that impaired stoichiometry between mtHsp40 and mtHsp70 promotes Opa1_L cleavage, leading to cristae opening, decreased OXPHOS, and triggering of mitochondrial fragmentation after reduction in their chaperone function.     

Mitochondrially localized active caspase-9 and caspase-3 result mostly from translocation from the cytosol and partly from caspase-mediated activation in the organelle. Lack of evidence for Apaf-1-mediated procaspase-9 activation in the mitochondria

Active caspase-9 and caspase-3 have been observed in the mitochondria, but their origins are unclear. Theoretically, procaspase-9 might be activated in the mitochondria in a cytochrome c/Apaf-1-dependent manner, or activated caspase-9 and -3 may translocate to the mitochondria, or the mitochondrially localized procaspases may be activated by the translocated active caspases. Here we present evidence that the mitochondrially localized active caspase-9 and -3 result mostly from translocation from the cytosol (into the intermembrane space) and partly from caspase-mediated activation in the organelle rather than from the Apaf-1-mediated activation. Apaf-1 localizes exclusively in the cytosol and, upon apoptotic stimulation, translocates to the perinuclear area but not to the mitochondria. In most cases, the mitochondrially localized procaspase-9 and -3 are released early during apoptosis and translocate to the cytosol and/or perinuclear area. Cytochrome c and the mitochondrial matrix protein Hsp60 are also rapidly released to the cytosol early during apoptosis. Both the early release of proteins like cytochrome c and Hsp60 from the mitochondria as well as the later translocation of the active caspase-9/-3 are partially inhibited by cyclosporin A, an inhibitor of mitochondrial membrane permeabilization. The mitochondrial active caspases may function as a positive feedback mechanism to further activate other or residual mitochondrial procaspases, degrade mitochondrial constituents, and disintegrate mitochondrial functions.     

Dual specificity phosphatases 18 and 21 target to opposing sides of the mitochondrial inner membrane

Although large-scale approaches have identified numerous mitochondrial phosphoproteins, little is known about the mitochondrial kinases and phosphatases that regulate these phosphoproteins. Here, we identify two members of the atypical dual specificity phosphatases (DSP), DSP18 and DSP21, that are localized in mitochondria. Although DSP18 is widely expressed in several mammalian tissues, DSP21 is selectively expressed in the testes. We demonstrate that DSP18 and DSP21 are targeted to mitochondria by cryptic internal localization signals. Subfractionation of mitochondria demonstrated that DSP18 is located in the intermembrane space as a peripheral membrane protein of the inner membrane. In contrast, subfractionation of rat testis mitochondria revealed DSP21 is localized to the matrix as a peripheral membrane protein of the inner membrane. Moreover, we demonstrate that a previously reported substrate for DSP18, the stress-activated protein kinase, does not localize to mitochondria in several different tissues, making it an unlikely substrate for DSP18. Finally, we show that induction of apoptosis by treatment with staurosporine causes translocation of DSP18 from the intermembrane space into the cytosol similar to other apoptogenic factors such as cytochrome c. This work rigorously demonstrates the unique location of two highly similar DSPs on opposing sides of the mitochondrial inner membrane.     

Bcl-2-family proteins and the role of mitochondria in apoptosis

Mitochondria are central to many forms of cell death, usually via the release of pro-apoptotic proteins from the mitochondrial intermembrane space. Some intermembrane space proteins, including cytochrome c, Smac/DIABLO, and Omi/Htra2, can induce or enhance caspase activation, whereas others, such as AIF and endonuclease G, might act in a caspase-independent manner. Intermembrane space protein release is often regulated by Bcl-2-family proteins. Recent evidence suggests that pro-apoptotic members of this family, by themselves, can permeabilize the outer mitochondrial membrane without otherwise damaging mitochondria. Mitochondria can contribute to cell death in other ways. For example, they can respond to calcium release from the endoplasmic reticulum by undergoing the mitochondrial permeability transition, which in turn causes outer membrane rupture and the release of intermembrane space proteins. Bcl-2-family proteins can influence the levels of releasable Ca(2+) in the endoplasmic reticulum, and thus determine whether the released Ca(2+) is sufficient to overload mitochondria and induce cell death.