Fatty Acid Synthase Impacts the Pathobiology of Candida parapsilosis In Vitro and during Mammalian Infection

Cytosolic fungal fatty acid synthase is composed of two subunits α and β, which are encoded by Fas1 and Fas2 genes. In this study, the Fas2 genes of the human pathogen Candida parapsilosis were deleted using a modified SAT1 flipper technique. CpFas2 was essential in media lacking exogenous fatty acids and the growth of Fas2 disruptants (Fas2 KO) was regulated by the supplementation of different long chain fatty acids, such as myristic acid (14∶0), palmitic acid (16∶0), and Tween 80, in a dose-specific manner. Lipidomic analysis revealed that Fas2 KO cells were severely restricted in production of unsaturated fatty acids. The Fas2 KO strains were unable to form normal biofilms and were more efficiently killed by murine-like macrophages, J774.16, than the wild type, heterozygous and reconstituted strains. Furthermore, Fas2 KO yeast were significantly less virulent in a systemic murine infection model. The Fas2 KO cells were also hypersensitive to human serum, and inhibition of CpFas2 in WT C. parapsilosis by cerulenin significantly decreased fungal growth in human serum. This study demonstrates that CpFas2 is essential for C. parapsilosis growth in the absence of exogenous fatty acids, is involved in unsaturated fatty acid production, influences fungal virulence, and represents a promising antifungal drug target.     

Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis: An essential role of fatty acid desaturase Ole1

Elevated levels of glucose and lipids can result in cellular dysfunction in eukaryotic cells ranging from Saccharomyces cerevisiae yeasts to human cells. Moreover, glucotoxicity and lipotoxicity can cause cell death, although the mechanism(s) for lethality is unclear. In the present study, we utilized Candida parapsilosis fatty acid desaturase (OLE1) and fatty acid synthase (FAS2) gene deletion mutants and wild-type (WT) yeast cells to unravel the relationship to glucose and lipid induced cell death in eukaryotic cells. Incubation of WT yeast cells with glucose led to the rapid accumulation of lipid droplets, whereas lipid droplet formation was severely impaired in yeast cells with deletion of OLE1 (ole1Δ/Δ) or FAS2 (fas2Δ/Δ). Interestingly, ole1Δ/Δ yeast cells died within hours in a 1% glucose medium without fatty acid supplementation, whereas the WT or fas2Δ/Δ yeast cells did not. In glucose medium, ole1Δ/Δ yeast cells accumulated saturated fatty acids, while fas2Δ/Δ did not. Addition of saturated fatty acids (e.g., palmitic acid) enhanced ole1Δ/Δ yeast cell death, whereas the addition of unsaturated fatty acids (e.g., oleic or palmitoleic acid) rescued cell death. Furthermore, palmitic acid and glucose medium induced apopotic cell death in ole1Δ/Δ yeast cells, which was dependent on mitochondrial function. Thus, our results show that glucotoxicity is directly linked to lipotoxicity, which we demonstrate is mediated by mitochondrial function.     

14-3-3 Protects against stress-induced apoptosis

Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3β/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3β/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3β/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3β/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called ‘autophagic death''. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3β/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins.     

Inhibition of peroxisome fission,but not mitochondrial fission,increases yeast chronological lifespan

Mitochondria are key players in aging and cell death. It has been suggested that mitochondrial fragmentation, mediated by the Dnm1/Fis1 organelle fission machinery, stimulates aging and cell death. This was based on the observation that Saccharomyces cerevisiae Δdnm1 and Δfis1 mutants show an enhanced lifespan and increased resistance to cell death inducers. However, the Dnm1/Fis1 fission machinery is also required for peroxisome division. Here we analyzed the significance of peroxisome fission in yeast chronological lifespan, using yeast strains in which fission of mitochondria was selectively blocked. Our data indicate that the lifespan extension caused by deletion of FIS1 is mainly due to a defect in peroxisome fission and not caused by a block in mitochondrial fragmentation. These observations are underlined by our observation that deletion of FIS1 does not lead to lifespan extension in yeast peroxisome deficient mutant cells.     

Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C20 Fatty Acids to C60-to-C90 Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C₂₀ fatty acids to form C₆₀-to-C₉₀ mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Δ cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Δ cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C₄ to C₈) than was previously thought (>C₁₂). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.     

Vital function of PRELI and essential requirement of its LEA motif

Proteins containing the late embryogenesis abundant (LEA) motif comprise a conserved family, postulated to act as cell protectors. However, their function and mechanisms of action remain unclear. Here we show that PRELI, a mammalian LEA-containing homolog of yeast Ups1p, can associate with dynamin-like GTPase Optic Atrophy-1 (OPA1) and contribute to the maintenance of mitochondrial morphology. Accordingly, PRELI can uphold mitochondrial membrane potential (ΔΨ_m) and enhance respiratory chain (RC) function, shown by its capacity to induce complex-I/NADH dehydrogenase and ATP synthase expression, increase oxygen consumption and reduce reactive oxygen species (ROS) production. PRELI can also inhibit cell death induced by STS, TNF-α or UV irradiation. Moreover, in vitro and in vivo dominant-negative overexpression of mutant PRELI/LEA⁻ (lacking the LEA motif) and transient in vitro PRELI-specific knockdown can render lymphocytes vulnerable to apoptosis, cause mouse embryo lethality and revert the resistance of lymphoma cells to induced death. Collectively, these data support the long-presumed notion of LEA protein-dependent mechanisms of cytoprotection and suggest that PRELI interacts with OPA1 to maintain mitochondria structures intact, sustain balanced ion⁻/proton⁺ gradients, promote oxidative phosphorylation reactions, regulate pro- and antiapoptotic protein traffic and enable cell responses to induced death. These findings may help to understand how bioenergetics is mechanistically connected with cell survival cues.     

A Fox2-Dependent Fatty Acid ?-Oxidation Pathway Coexists Both in Peroxisomes and Mitochondria of the Ascomycete Yeast Candida lusitaniae

It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying ¹⁴C_α-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner.     

Protection of pancreatic INS-1 ��-cells from glucose- and fructose-induced cell death by inhibiting mitochondrial permeability transition with cyclosporin A or metformin

Hyperglycemia is detrimental to β-cell viability, playing a major role in the progression of β-cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. Recent evidence suggests that PTP inhibitors prevent hyperglycemia-induced cell death in human endothelial cells. In this work, we have examined the involvement of PTP opening in INS-1 cell death induced by high levels of glucose or fructose. PTP regulation was studied by measuring the calcium retention capacity in permeabilized INS-1 cells and by confocal microscopy in intact INS-1 cells. Cell death was analyzed by flow cytometry. We first reported that metformin and cyclosporin A (CsA) prevented Ca²⁺-induced PTP opening in permeabilized and intact INS-1 cells. We then showed that incubation of INS-1 cells in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. As both metformin and CsA prevented glucose- and fructose- induced PTP opening, and hampered glucose- and fructose- induced cell death, we conclude that PTP opening is involved in high glucose- and high fructose- induced INS-1 cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β-cell viability.     

Reversible Cyclosporin A-sensitive Mitochondrial Depolarization Occurs within Minutes of Stroke Onset in Mouse Somatosensory Cortex in Vivo: A TWO-PHOTON IMAGING STUDY*

Neuronal structure and function are rapidly damaged during global ischemia but can in part recover during reperfusion. Despite apparent recovery in the hours/days following an ischemic episode, delayed cell death can be initiated, making it important to understand how initial ischemic events affect potential mediators of apoptosis. Mitochondrial dysfunction and the opening of the mitochondrial permeability transition pore (mPTP) are proposed to link ischemic ionic imbalance to mitochondrially mediated cell death pathways. Using two-photon microscopy, we monitored mitochondrial transmembrane potential (Δψ_m) in vivo within the somatosensory cortex during ischemia and reperfusion in a mouse global ischemia model. Our results indicated a synchronous loss of Δψ_m within 1–3 min of ischemic onset that was linked to within seconds of plasma membrane potential (Δψ_p) depolarization. Δψ_m recovered rapidly upon reperfusion, and no delayed depolarization was observed over 2 h. Cyclosporin A treatment largely blocked Δψ_m collapse during ischemia, suggesting a role for the mPTP. Blocking Δψ_m depolarization did not affect structural damage to dendrites, indicating that the opening of the mPTP and damage to dendrites are separable pathways that are activated during Δψ_p depolarization. Our findings using in vivo imaging suggest that mitochondrial dysfunction and specifically the activation of the mPTP are early reversible events during brain ischemia that could trigger delayed cell death.     

The function of mitochondria in presynaptic development at the neuromuscular junction

Mitochondria with high membrane potential (ΔΨ_m) are enriched in the presynaptic nerve terminal at vertebrate neuromuscular junctions, but the exact function of these localized synaptic mitochondria remains unclear. Here, we investigated the correlation between mitochondrial ΔΨ_m and the development of synaptic specializations. Using mitochondrial ΔΨ_m-sensitive probe JC-1, we found that ΔΨ_m in Xenopus spinal neurons could be reversibly elevated by creatine and suppressed by FCCP. Along naïve neurites, preexisting synaptic vesicle (SV) clusters were positively correlated with mitochondrial ΔΨ_m, suggesting a potential regulatory role of mitochondrial activity in synaptogenesis. Indicating a specific role of mitochondrial activity in presynaptic development, mitochondrial ATP synthase inhibitor oligomycin, but not mitochondrial Na⁺/Ca²⁺ exchanger inhibitor CGP-37157, inhibited the clustering of SVs induced by growth factor–coated beads. Local F-actin assembly induced along spinal neurites by beads was suppressed by FCCP or oligomycin. Our results suggest that a key role of presynaptic mitochondria is to provide ATP for the assembly of actin cytoskeleton involved in the assembly of the presynaptic specialization including the clustering of SVs and mitochondria themselves.     

FADS2 function loss at the cancer hotspot 11q13 locus diverts lipid signaling precursor synthesis to unusual eicosanoid fatty acids

Background

Genes coding for the fatty acid desaturases (FADS1, 2, 3) localized at the cancer genomic hotspot 11q13 locus are required for the biosynthesis of 20 carbon polyunsaturated fatty acids (PUFA) that are direct eicosanoid precursors. In several cancer cell lines, FADS2 encoded Δ6 and Δ8 desaturation is not functional.

Methodology/Principal Findings

Analyzing MCF7 cell fatty acids with detailed structural mass spectrometry, we show that in the absence of FADS2 activity, the FADS1 product Δ5-desaturase operates to produce 5,11,14–20∶3 and 5,11,14,17–20∶4. These PUFA are missing the 8–9 double bond of the eicosanoid signaling precursors arachidonic acid (5,8,11,14–20∶4) and eicosapentaenoic acid (5,8,11,14,17–20∶5). Heterologous expression of FADS2 restores Δ6 and Δ8-desaturase activity and normal eicosanoid precursor synthesis.

Conclusions/Significance

The loss of FADS2-encoded activities in cancer cells shuts down normal PUFA biosynthesis, deleting the endogenous supply of eicosanoid and downstream docosanoid precursors, and replacing them with unusual butylene-interrupted fatty acids. If recapitulated in vivo, the normal eicosanoid and docosanoid cell signaling milieu would be depleted and altered due to reduction and substitution of normal substrates with unusual substrates, with unpredictable consequences for cellular communication.     

Role of the mitochondria in immune-mediated apoptotic death of the human pancreatic β cell line βLox5

Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA) depleted βLox5 cells, or βLox5 ρ(0) cells. βLox5 ρ(0) cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ(0) cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.     

Role of Pex11p in Lipid Homeostasis in Yarrowia lipolytica

Peroxisomes are essential organelles in the cells of most eukaryotes, from yeasts to mammals. Their role in β-oxidation is particularly essential in yeasts; for example, in Saccharomyces cerevisiae, fatty acid oxidation takes place solely in peroxisomes. In this species, peroxisome biogenesis occurs when lipids are present in the culture medium, and it involves the Pex11p protein family: ScPex11p, ScPex25p, ScPex27p, and ScPex34p. Yarrowia lipolytica has three Pex11p homologues, which are YALI0C04092p (YlPex11p), YALI0C04565p (YlPex11C), and YALI0D25498p (Pex11/25p). We found that these genes are regulated by oleic acid, and as has been observed in other organisms, YlPEX11 deletion generated giant peroxisomes when mutant yeast were grown in oleic acid medium. Moreover, ΔYlpex11 was unable to grow on fatty acid medium and showed extreme dose-dependent sensitivity to oleic acid. Indeed, when the strain was grown in minimum medium with 0.5% glucose and 3% oleic acid, lipid body lysis and cell death were observed. Cell death and lipid body lysis may be partially explained by an imbalance in the expression of the genes involved in lipid storage, namely, DGA1, DGA2, and LRO1, as well as that of TGL4, which is involved in lipid remobilization. TGL4 deletion and DGA2 overexpression resulted in decreased oleic acid sensitivity and delayed cell death of ΔYlpex11, which probably stemmed from the release of free fatty acids into the cytoplasm. All these results show that YlPex11p plays an important role in lipid homeostasis in Y. lipolytica.     

Mitochondrial dysfunction links ceramide activated HRK expression and cell death

Purpose

Cell death is an essential process in normal development and homeostasis. In eyes, corneal epithelial injury leads to the death of cells in underlying stroma, an event believed to initiate corneal wound healing. The molecular basis of wound induced corneal stromal cell death is not understood in detail. Studies of others have indicated that ceramide may play significant role in stromal cell death following LASIK surgery. We have undertaken the present study to investigate the mechanism of death induced by C6 ceramide in cultures of human corneal stromal (HCSF) fibroblasts.

Methods

Cultures of HCSF were established from freshly excised corneas. Cell death was induced in low passage (p<4) cultures of HCSF by treating the cells with C6 ceramide or C6 dihydroceramide as a control. Cell death was assessed by Live/Dead cell staining with calcein AM and ethidium homodimer-1 as well as Annexin V staining, caspase activation and TUNEL staining Mitochondrial dysfunction was assessed by Mito Sox Red, JC-1 and cytochrome C release Gene expression was examined by qPCR and western blotting.

Results

Our data demonstrate ceramide caused mitochondrial dysfunction as evident from reduced MTT staining, cyto c release from mitochondria, enhanced generation of ROS, and loss in mitochondrial membrane potential (ΔΨ_m). Cell death was evident from Live -Dead Cell staining and the inability to reestablish cultures from detached cells. Ceramide induced the expression of the harikari gene(HRK) and up-regulated JNK phosphorylation. In ceramide treated cells HRK was translocated to mitochondria, where it was found to interact with mitochondrial protein p32. The data also demonstrated HRK, p32 and BAD interaction. Ceramide-induced expression of HRK, mitochondrial dysfunction and cell death were reduced by HRK knockdown with HRK siRNA.

Conclusion

Our data document that ceramide is capable of inducing death of corneal stromal fibroblasts through the induction of HRK mediated mitochondria dysfunction.     

A C. elegans Model for Mitochondrial Fatty Acid Synthase II: The Longevity-Associated Gene W09H1.5/mecr-1 Encodes a 2-trans-Enoyl-Thioester Reductase

Our recognition of the mitochondria as being important sites of fatty acid biosynthesis is continuously unfolding, especially in light of new data becoming available on compromised fatty acid synthase type 2 (FASII) in mammals. For example, perturbed regulation of murine 17β-HSD8 encoding a component of the mitochondrial FASII enzyme 3-oxoacyl-thioester reductase is implicated in polycystic kidney disease. In addition, over-expression in mice of the Mecr gene coding for 2-trans-enoyl-thioester reductase, also of mitochondrial FASII, leads to impaired heart function. However, mouse knockouts for mitochondrial FASII have hitherto not been reported and, hence, there is a need to develop alternate metazoan models such as nematodes or fruit flies. Here, the identification of Caenorhabditis elegans W09H1.5/MECR-1 as a 2-trans-enoyl-thioester reductase of mitochondrial FASII is reported. To identify MECR-1, Saccharomyces cerevisiae etr1Δ mutant cells were employed that are devoid of mitochondrial 2-trans-enoyl-thioester reductase Etr1p. These yeast mutants fail to synthesize sufficient levels of lipoic acid or form cytochrome complexes, and cannot respire or grow on non-fermentable carbon sources. A mutant yeast strain ectopically expressing nematode mecr-1 was shown to contain reductase activity and resemble the self-complemented mutant strain for these phenotype characteristics. Since MECR-1 was not intentionally targeted for compartmentalization using a yeast mitochondrial leader sequence, this inferred that the protein represented a physiologically functional mitochondrial 2-trans-enoyl-thioester reductase. In accordance with published findings, RNAi-mediated knockdown of mecr-1 in C. elegans resulted in life span extension, presumably due to mitochondrial dysfunction. Moreover, old mecr-1(RNAi) worms had better internal organ appearance and were more mobile than control worms, indicating a reduced physiological age. This is the first report on RNAi work dedicated specifically to curtailing mitochondrial FASII in metazoans. The availability of affected survivors will help to position C. elegans as an excellent model for future pursuits in the emerging field of mitochondrial FASII research.     

Effects of physiological manipulation on the kinetics of mitochondrial phosphate transport in Saccharomyces cerevisiae

The kinetics of [³²P]phosphate uptake has been studied in different types of Saccharomyces cerevisiae mitochondria. Mitochondria were isolated from yeast grown aerobically on 2% lactate (Lac-mitochondria), 2% galactose (Gal-mitochondria), 5.4% glucose (Glu-mitochondria) or from yeast grown anaerobically on 2% galactose (Promitochondria). The effect of chloramphenicol was also studied by adding it to the growth medium of yeast grown aerobically on 2% galactose (chloramphenicol-mitochondria).[³²P]Phosphate uptake followed an oscillatory pattern in Lac, Gal-mitochondria and Promitochondria.Saturation kinetics were detected in fully differenciated mitochondria and in Promitochondria, but not in chloramphenicol-mitochondria.Glu-mitochondria did not translocate phosphate as shown both by lack of [³²P]phosphate uptake and lack of swelling in isoosmotic potassium solution.Repressed yeast cells were incubated in a resting cell medium and mitochondria were isolated at different times of incubation. The rate of respiration and the oligomycin-sensitive ATPase increased during the course of the incubation. After 2h, a mitochondrial mersalyl-sensitive swelling in an isoosmotic potassium phosphate solution was detected.As expected, no increase of the rate of respiration was observed when chloramphenicol was added in the derepression medium. But the oligomycin-sensitive ATPase decreased. Chloramphenicol did not affect the phosphate transport activity as measured by the swelling of mitochondria, but the [³²P]phosphate uptake did not follow saturation kinetics. A complete derepression of the inorganic phosphate-carrier activity was achieved by a 4 h incubation of the repressed cells in the presence of chloramphenicol, followed by a 6 h incubation in presence of cycloheximide.These data strongly suggest that the mitochondrial protein-synthesis system is required for the normal function of the inorganic phosphate-carrier.