Mitochondria-specific Lipid Research Articles

MG53 preserves mitochondrial integrity of cardiomyocytes during ischemia reperfusion-induced oxidative stress

Ischemic injury to the heart induces mitochondrial dysfunction due to increasing oxidative stress. MG53, also known as TRIM72, is highly expressed in striated muscle, is secreted as a myokine after exercise, and is essential for repairing damaged plasma membrane of many tissues by interacting with the membrane lipid phosphatidylserine (PS). We hypothesized MG53 could preserve mitochondrial integrity after an ischemic event by binding to the mitochondrial-specific lipid, cardiolipin (CL), for mitochondria protection to prevent mitophagy. Fluorescent imaging and Western blotting experiments showed recombinant human MG53 (rhMG53) translocated to the mitochondria after ischemic injury in vivo and in vitro. Fluorescent imaging indicated rhMG53 treatment reduced superoxide generation in ex vivo and in vitro models. Lipid-binding assay indicated MG53 binds to CL. Transfecting cardiomyocytes with the mitochondria-targeted mt-mKeima showed inhibition of mitophagy after MG53 treatment. Overall, we show that rhMG53 treatment may preserve cardiac function by preserving mitochondria in cardiomyocytes. These findings suggest MG53's interactions with mitochondria could be an attractive avenue for developing MG53 as a targeted protein therapy for cardioprotection.

Abstract 13835: MG53 Preserves Mitochondrial Integrity of Cardiomyocytes in Pre-Clinical Models of Ischemia Reperfusion-Induced Oxidative Stress

Introduction: Ischemic injury to the heart induces mitochondrial dysfunction due to an increase in oxidative stress. MG53, also known as TRIM72, is highly expressed in striated muscle and is essential for repairing damaged plasma membranes by interacting with the membrane lipid phosphatidylserine. We have previously shown that mg53-/- mice are more susceptible to ischemia-reperfusion injury, and treatment with exogenous recombinant human MG53 (rhMG53) reduces infarct damage, restores cardiac function and preserves mitochondrial membrane potential. Hypothesis: We hypothesize MG53 preserves mitochondrial integrity after an ischemic event by binding to the mitochondrial-specific lipid, cardiolipin (CL), for mitochondrial membrane repair, preventing the formation of superoxides and mitophagy. Methods: Fluorescent imaging and western blotting experiments were conducted after ischemic injury in vivo and in vitro to measure the formation of superoxides and activation of mitophagy. A lipid binding assay was used to determine MG53’s ability to bind to CL. Transfection of murine cardiomyocyte cells with the mitochondria-targeted mt-mKeima was used to measure engulfment of the mitochondria by the lysosome. Results: We found that recombinant human MG53 (rhMG53) delivered prior ischemia/reperfusion translocated to the mitochondria both in vivo and in vitro. Additionally, superoxide generation was reduced by rhMG53 treatment using ex vivo and in vitro models. A lipid binding assay revealed rhMG53 binds to CL. Treating with rhMG53 inhibited mitophagy after ischemia/reperfusion, as demonstrated by the reduction of mitochondria in the lysosome after rhMG53 treatment and a reduction in mitochondria-bound parkin, a key mediator of mitophagy. Conclusions: In this study we provide evidence that rhMG53 treatment may preserve cardiac function by preserving mitochondria in cardiomyocytes. These findings suggest MG53’s interactions with mitochondria could be an attractive avenue for the development of MG53 as a targeted protein therapy for cardioprotection.

Interplay between cardiolipin and plasmalogens in Barth syndrome.

Barth syndrome (BTHS) is a rare inherited metabolic disease resulting from mutations in the gene of the enzyme tafazzin, which catalyzes the acyl chain remodeling of the mitochondrial-specific lipid cardiolipin (CL). Tissue samples of individuals with BTHS present abnormalities in the level and the molecular species of CL. In addition, in tissues of a tafazzin knockdown mouse as well as in cells derived from BTHS patients it has been shown that plasmalogens, a subclass of glycerophospholipids, also have abnormal levels. Likewise, administration of a plasmalogen precursor to cells derived from BTHS patients led to an increase in plasmalogen and to some extent CL levels. These results indicate an interplay between CL and plasmalogens in BTHS. This interdependence is supported by the concomitant loss in these lipids in different pathological conditions. However, currently the molecular mechanism linking CL and plasmalogens is not fully understood. Here, a review of the evidence showing the linkage between the levels of CL and plasmalogens is presented. In addition, putative mechanisms that might play a role in this interplay are proposed. Finally, the opportunity of therapeutic approaches based on the regulation of plasmalogens as new therapies for the treatment of BTHS is discussed.

Adipose lipidomics and RNA-Seq analysis revealed the enhanced mitochondrial function in UCP1 knock-in pigs

Uncoupling protein 1 (UCP1) plays a key role in nonshivering thermogenesis and is involved in the pathogenesis of obesity. In a previous study, we generated adipocyte-specific UCP1 knock-in (UCP1-KI) pigs, which exhibited improved thermoregulatory ability and decreased fat deposition. To investigate whether UCP1 knock-in alters the lipid composition of adipose tissues, lipidomics of inguinal subcutaneous white adipose tissue (iWAT) and backfat from 6-month-old cold-treated UCP1-KI pigs and wild-type (WT) pigs were profiled. In addition, genome-wide RNA-sequencing of iWAT was performed to further study the genetic basis for lipid alterations. The results showed that iWAT and backfat from UCP1-KI pigs exhibited distinct lipidomic profiles, as the mild lipid alteration was observed in backfat of UCP1 knock-in pigs. Inguinal WAT from UCP1-KI pigs contained significantly decreased total triacylglycerol (p < 0.05), together with the downregulation of genes involved in fatty acid metabolism, suggesting the decreased lipogenesis in iWAT of UCP1-KI pigs. Significantly increased levels of total sphingolipids (p<0.05) were also observed in iWAT from UCP1-KI pigs. Notably, two mitochondrial-specific lipid species, cardiolipin CL72:8 (18:2) and CL74:9 (18:2), were found to be dramatically increased in iWAT from UCP1-KI pigs, suggesting enhanced mitochondrial function. This observation was further supported by the significant upregulation of numerous mitochondrial-related genes and significantly increased number of large mitochondria and mitochondrial cristae in iWAT of UCP1-KI pigs. Taken together, these data illustrate the specific role of UCP1 in lipid metabolism of fat tissues in pigs and provide new data for characterization of fat traits in UCP1-KI pigs.

Mitochondria Released by Apoptotic Cell Death Initiate Innate Immune Responses.

In solid organ transplantation, cell death arising from ischemia/reperfusion leads to the release of several damage-associated molecular patterns derived from mitochondria. Mitochondrial damage-associated molecular patterns (mtDAMPs) initiate proinflammatory responses, but it remains unknown whether the mode of cell death affects the inflammatory properties of mitochondria. Murine and human cell lines induced to selectively undergo apoptosis and necroptosis were used to examine the extracellular release of mitochondria during programmed cell death. Mitochondria purified from healthy, apoptotic, and necroptotic cells were used to stimulate macrophage inflammasome responses in vitro and neutrophil chemotaxis in vivo. Inhibition of specific mtDAMPs was performed to identify those responsible for macrophage inflammasome activation. A rat liver transplant model was used to identify apoptotic and necroptotic cell death in graft tissue following ischemia/reperfusion. Both apoptotic and necroptotic cell death occur in parallel in graft tissue. Apoptotic cells released more mitochondria than necroptotic cells. Moreover, mitochondria from apoptotic cells were significantly more inflammatory in terms of macrophage inflammasome activation and neutrophil recruitment. Inhibition of cellular synthesis of cardiolipin, a mitochondria-specific lipid and mtDAMP, significantly reduced the inflammasome-activating properties of apoptosis-derived mitochondria. Mitochondria derived from apoptotic cells are potent activators of innate immune responses, whereas mitochondria derived from healthy or necroptotic cells are significantly less inflammatory. Cardiolipin appears to be a key mtDAMP-regulating inflammasome activation by mitochondria. Methods of inhibiting apoptotic cell death in transplant grafts may be beneficial for reducing graft inflammation and transplant allosensitization.

P 006 - Cytochrome c- cardiolipin complex: from peroxidase to Fenton chemistry

The small heme protein cytochrome c (cyt c) has important functions as redox carrier in the mitochondria. While located in the intermembrane space, it is closely associated with the mitochondria-specific lipid cardiolipin (CL). Upon release from stressed mitochondria, cyt c is also involved in apoptosis induction. However, in functioning mitochondria the physiological significance of alterations in cyt c-CL interaction remains largely unknown. Our studies have demonstrated a hydrogen peroxide-dependent iron release from cyt c, which resulted in “Fenton-like” lipid modifications in CL-containing liposomes in the presence of reducing agents, like ascorbic acid or glutathione. By using HPTLC and MALDI-TOF MS we could identify phosphatidic acid and phosphatidylhydroxyacetone as the fragmentation products of free radical transformations of the polar head group of cardiolipin. Moreover, in the hydrophobic part, we observed the formation of CL aldehydes, lyso-CL and lyso-CL aldehydes. The named products could have physiological significance as phosphatidic acid is a secondary messenger in mitochondria, while lyso-CL is involved in the reactions with the apoptosis-related protein tBid. Therefore, our results may suggest a non-enzymatic pathway for the production of CL-derived biologically active compounds.

High-affinity cooperative Ca2+ binding by MICU1-MICU2 serves as an on-off switch for theuniporter.

The mitochondrial calcium uniporter is a Ca2+-activated Ca2+ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca2+ signals. It is regulated by two paralogous EF-hand proteins-MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca2+ We reconstitute the MICU1-MICU2 heterodimer and demonstrate that it binds Ca2+ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria-specific lipid cardiolipin. We determine the minimum Ca2+ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca2+-binding affinity of MICU1-MICU2. We conclude that cooperative, high-affinity interaction of the MICU1-MICU2 complex with Ca2+ serves as an on-off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca2+ signals.

Purity matters: A workflow for the valid high-resolution lipid profiling of mitochondria from cell culture samples.

Subcellular lipidomics is a novel field of research that requires the careful combination of several pre-analytical and analytical steps. To define a reliable strategy for mitochondrial lipid profiling, we performed a systematic comparison of different mitochondria isolation procedures by western blot analyses and comprehensive high-resolution lipidomics. Using liver-derived HepG2 cells, we compared three common mitochondria isolation methods, differential centrifugation (DC), ultracentrifugation (UC) and a magnetic bead-assisted method (MACS). In total, 397 lipid species, including 32 cardiolipins, could be quantified in only 100 μg (by protein) of purified mitochondria. Mitochondria isolated by UC showed the highest enrichment in the mitochondria-specific cardiolipins as well as their precursors, phosphatidylglycerols. Mitochondrial fractions obtained by the commonly used DC and the more recent MACS method contained substantial contaminations by other organelles. Employing these isolation methods when performing lipidomics analyses from cell culture mitochondria may lead to inaccurate results. To conclude, we present a protocol how to obtain reliable mitochondria-specific lipid profiles from cell culture samples and show that quality controls are indispensable when performing mitochondria lipidomics.

Complex I and complex III inhibition specifically increase cytosolic hydrogen peroxide levels without inducing oxidative stress in HEK293 cells

Inhibitor studies with isolated mitochondria demonstrated that complex I (CI) and III (CIII) of the electron transport chain (ETC) can act as relevant sources of mitochondrial reactive oxygen species (ROS). Here we studied ROS generation and oxidative stress induction during chronic (24h) inhibition of CI and CIII using rotenone (ROT) and antimycin A (AA), respectively, in intact HEK293 cells. Both inhibitors stimulated oxidation of the ROS sensor hydroethidine (HEt) and increased mitochondrial NAD(P)H levels without major effects on cell viability. Integrated analysis of cells stably expressing cytosolic- or mitochondria-targeted variants of the reporter molecules HyPer (H2O2-sensitive and pH-sensitive) and SypHer (H2O2-insensitive and pH-sensitive), revealed that CI- and CIII inhibition increased cytosolic but not mitochondrial H2O2 levels. Total and mitochondria-specific lipid peroxidation was not increased in the inhibited cells as reported by the C11-BODIPY581/591 and MitoPerOx biosensors. Also expression of the superoxide-detoxifying enzymes CuZnSOD (cytosolic) and MnSOD (mitochondrial) was not affected. Oxyblot analysis revealed that protein carbonylation was not stimulated by CI and CIII inhibition. Our findings suggest that chronic inhibition of CI and CIII: (i) increases the levels of HEt-oxidizing ROS and (ii) specifically elevates cytosolic but not mitochondrial H2O2 levels, (iii) does not induce oxidative stress or substantial cell death. We conclude that the increased ROS levels are below the stress-inducing level and might play a role in redox signaling.

PII: S0891-5849(15)00979-X

Ischemic injury to the heart induces mitochondrial dysfunction due to increasing oxidative stress. MG53, also known as TRIM72, is highly expressed in striated muscle, is secreted as a myokine after exercise, and is essential for repairing damaged plasma membrane of many tissues by interacting with the membrane lipid phosphatidylserine (PS). We hypothesized MG53 could preserve mitochondrial integrity after an ischemic event by binding to the mitochondrial-specific lipid, cardiolipin (CL), for mitochondria protection to prevent mitophagy. Fluorescent imaging and Western blotting experiments showed recombinant human MG53 (rhMG53) translocated to the mitochondria after ischemic injury in vivo and in vitro. Fluorescent imaging indicated rhMG53 treatment reduced superoxide generation in ex vivo and in vitro models. Lipid-binding assay indicated MG53 binds to CL. Transfecting cardiomyocytes with the mitochondria-targeted mt-mKeima showed inhibition of mitophagy after MG53 treatment. Overall, we show that rhMG53 treatment may preserve cardiac function by preserving mitochondria in cardiomyocytes. These findings suggest MG53's interactions with mitochondria could be an attractive avenue for developing MG53 as a targeted protein therapy for cardioprotection.

Methodology for use of mitochondria-targeted cations in the field of oxidative stress-related research.

For many pathological conditions, reactive oxygen species (ROS) generated in mitochondria are considered to have a role as a trigger. When mitochondrial ROS (mROS) are formed in the inner mitochondrial membrane, they initiate free radical-mediated chain reactions of lipid peroxidation and are thus especially damaging. The consequences of membrane damage are decreased electrical resistance of the membrane, oxidative damage to cardiolipin (a mitochondria specific lipid essential for functioning of respiratory chain proteins and H(+)-ATP synthase), and damage to mitochondrial DNA localized in close vicinity to the inner membrane, with consequent mitochondrial dysfunction and induction of apoptotic cascade and cell death. To target the starting point of such undesirable events, antioxidants conjugated with mitochondria-targeted, membrane-penetrating cations can be used to scavenge ROS inside mitochondria. The most demonstrative indications favoring this conclusion originate from recent discoveries of the in vivo effects of such cations belonging to the MitoQ and SkQ groups. Here we describe some essential methodological aspects of the application of mitochondria-targeted cations promising in treating oxidative stress-related pathologies.

A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission.

The GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial division, but the mechanisms remain poorly understood. Much of what is attributed to Drp1's mechanism of action in mitochondrial membrane fission parallels that of prototypical dynamin in endocytic vesicle scission. Unlike the case for dynamin, however, no lipid target for Drp1 activation at the mitochondria has been identified. In addition, the oligomerization properties of Drp1 have not been well established. We show that the mitochondria-specific lipid cardiolipin is a potent stimulator of Drp1 GTPase activity, as well as of membrane tubulation. We establish further that under physiological conditions, Drp1 coexists as two morphologically distinct polymeric species, one nucleotide bound in solution and the other membrane associated, which equilibrate via a dimeric assembly intermediate. With two mutations, C300A and C505A, that shift Drp1 polymerization equilibria in opposite directions, we demonstrate that dimers, and not multimers, potentiate the reassembly and reorganization of Drp1 for mitochondrial membrane remodeling both in vitro and in vivo.

E3 Ligase Subunit Fbxo15 and PINK1 Kinase Regulate Cardiolipin Synthase 1 Stability and Mitochondrial Function in Pneumonia

SUMMARYAcute lung injury (ALI) is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1). Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme.

Characterization of cardiolipins and their oxidation products by LC–MS analysis

Cardiolipins, a class of mitochondria-specific lipid molecules, is one of the most unusual and ancient phospholipids found in essentially all living species. Typical of mammalian cells is the presence of vulnerable to oxidation polyunsaturated fatty acid resides in CL molecules. The overall role and involvement of cardiolipin oxidation (CLox) products in major intracellular signaling as well as extracellular inflammatory and immune responses have been established. However, identification of individual peroxidized molecular species in the context of their ability to induce specific biological responses has not been yet achieved. This is due, at least in part, to technological difficulties in detection, identification, structural characterization and quantitation of CLox associated with their very low abundance and exquisite diversification. This dictates the need for the development of new methodologies for reliable, sensitive and selective analysis of both CLox. LC–MS-based oxidative lipidomics with high mass accuracy instrumentation as well as new software packages are promising in achieving the goals of expedited and reliable analysis of cardiolipin oxygenated species in biosamples.

Deacylation on the matrix side of the mitochondrial inner membrane regulates cardiolipin remodeling

The mitochondrial-specific lipid cardiolipin (CL) is required for numerous processes therein. After its synthesis on the matrix-facing leaflet of the inner membrane (IM), CL undergoes acyl chain remodeling to achieve its final form. In yeast, this process is completed by the transacylase tafazzin, which associates with intermembrane space (IMS)-facing membrane leaflets. Mutations in TAZ1 result in the X-linked cardiomyopathy Barth syndrome. Amazingly, despite this clear pathophysiological association, the physiological importance of CL remodeling is unresolved. In this paper, we show that the lipase initiating CL remodeling, Cld1p, is associated with the matrix-facing leaflet of the mitochondrial IM. Thus monolysocardiolipin generated by Cld1p must be transported to IMS-facing membrane leaflets to gain access to tafazzin, identifying a previously unknown step required for CL remodeling. Additionally, we show that Cld1p is the major site of regulation in CL remodeling; and that, like CL biosynthesis, CL remodeling is augmented in growth conditions requiring mitochondrially produced energy. However, unlike CL biosynthesis, dissipation of the mitochondrial membrane potential stimulates CL remodeling, identifying a novel feedback mechanism linking CL remodeling to oxidative phosphorylation capacity.

A Mathematical Model for the Determination of Steady-State Cardiolipin Remodeling Mechanisms Using Lipidomic Data

Technical advances in lipidomic analysis have generated tremendous amounts of quantitative lipid molecular species data, whose value has not been fully explored. We describe a novel computational method to infer mechanisms of de novo lipid synthesis and remodeling from lipidomic data. We focus on the mitochondrial-specific lipid cardiolipin (CL), a polyglycerol phospholipid with four acyl chains. The lengths and degree of unsaturation of these acyl chains vary across CL molecules, and regulation of these differences is important for mitochondrial energy metabolism. We developed a novel mathematical approach to determine mechanisms controlling the steady-state distribution of acyl chain combinations in CL . We analyzed mitochondrial lipids from 18 types of steady-state samples, each with at least 3 replicates, from mouse brain, heart, lung, liver, tumor cells, and tumors grown in vitro. Using a mathematical model for the CL remodeling mechanisms and a maximum likelihood approach to infer parameters, we found that for most samples the four chain positions have an independent and identical distribution, indicating they are remodeled by the same processes. Furthermore, for most brain samples and liver, the distribution of acyl chains is well-fit by a simple linear combination of the pools of acyl chains in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG). This suggests that headgroup chemistry is the key determinant of acyl donation into CL, with chain length/saturation less important. This canonical remodeling behavior appears damaged in some tumor samples, which display a consistent excess of CL molecules having particular masses. For heart and lung, the “proportional incorporation” assumption is not adequate to explain the CL distribution, suggesting additional acyl CoA-dependent remodeling that is chain-type specific. Our findings indicate that CL remodeling processes can be described by a small set of quantitative relationships, and that bioinformatic approaches can help determine these processes from high-throughput lipidomic data.

Single vesicle observations of the cardiolipin-cytochrome C interaction: induction of membrane morphology changes.

We present a novel platform for investigating the composition-specific interactions of proteins (or other biologically relevant molecules) with model membranes composed of compositionally distinct domains. We focus on the interaction between a mitochondrial-specific lipid, cardiolipin (CL), and a peripheral membrane protein, cytochrome c (cyt c). We engineer vesicles with compositions such that they phase separate into coexisting liquid phases and the lipid of interest, CL, preferentially localizes into one of the domains (the liquid disordered (L(d)) phase). The presence of CL-rich and CL-depleted domains within the same vesicle provides a built-in control experiment to simultaneously observe the behavior of two membrane compositions under identical conditions. We find that cyt c binds strongly to CL-rich domains and observe fascinating morphological transitions within these regions of membrane. CL-rich domains start to form small buds and eventually fold up into a collapsed state. We also observe that cyt c can induce a strong attraction between the CL-rich domains of adjacent vesicles as demonstrated by the development of large osculating regions between these domains. Qualitatively similar behavior is observed when other polycationic proteins or polymers of a similar size and net charge are used instead of cyt c. We argue that these striking phenomena can be simply understood by consideration of colloidal forces between the protein and the membrane. We discuss the possible biological implications of our observations in relation to the structure and function of mitochondria.

PiRNAs Meet Mitochondria

zuc/MitoPLD encodes a conserved enzyme that localizes to mitochondria and hydrolyzes the mitochondria-specific lipid cardiolipin. Surprisingly, zuc/MitoPLD activity is required for Piwi-interacting RNA (piRNA)-mediated silencing of transposable elements in fly and mouse germlines, suggesting that signaling from mitochondria influences the piRNA pathway.

Modulation of Membrane-Bound Glutathione Transferase Activity by Phospholipids Including Cardiolipin

Membrane-bound glutathione transferases (MGST1) distributed mostly in liver microsomal and mitochondrial membranes are activated by the thiol modification. In the present study, the effect of phospholipids on MGST1 activity was investigated using purified enzyme. When MGST1 was mixed with liposomes of cardiolipin (CL), phosphatidylcholine (PC), phosphatidylserine (PC), or phosphatidylethanolamine (PE), its activity was increased in a magnitude which was dependent on the anionic property of lipids in the order of CL>PS>PE>PC, indicating that MGST1 activity is enhanced by surrounding anionic lipids. Although MGST1 was activated by the thiol alkylation with N-ethylmaleimide (NEM), the activation was suppressed in the presence of anionic phospholipids as clearly observed in the presence of CL. Similarly, the activation of MGST1 by diamide or diamide plus glutathione through disulfide-bond formation was also disturbed in the presence of CL. Suppression of NEM-derived MGST1 activation by CL was lost when MGST1 was incubated with CL in the presence of the detergent Triton X-100. These results indicate that reactivity (stability) of the thiol in MGST1 is affected by surrounding lipids, namely CL which prevents MGST1 activation by thiol modification. Since CL is a mitochondria specific lipid located in the inner membrane, it was suggested that function of mitochondrial MGST1 could be regulated by interaction with CL.

Ups delivery to the intermembrane space of mitochondria: a novel affinity-driven protein import pathway

Mitochondria are comprised of two aqueous subcompartments: the matrix and the intermembrane space (IMS). Following their synthesis on cytosolic ribosomes, proteins destined to the matrix are threaded by N‐terminal matrix‐targeting sequences through TOM and TIM translocases in the outer and the inner membrane of the organelle. Proteins of the IMS often lack such presequences, and the principles mediating their uptake into mitochondria are diverse and poorly understood. In this issue, [Potting et al (2010)][1] and [Tamura et al (2010)][2] show that Ups proteins, a group of soluble IMS proteins critical for lipid homeostasis, adsorb to the IMS factor Mdm35 after their translocation across the outer membrane. Mdm35 thereby apparently functions as an intramitochondrial acceptor protein. The existence of affinity‐driven import routes has been proposed before, but Mdm35 represents the first intramitochondrial acceptor protein that is identified. [1]: #ref-9 [2]: #ref-12