Cellular Cholesterol Storage in the Niemann-Pick Disease Type C Mouse Is Associated with Increased Expression and Defective Processing of Apolipoprotein D

Abstract: Apolipoprotein D (apoD), a member of the lipocalin superfamily of ligand transporters, has been implicated in the transport of several small hydrophobic molecules including sterols and steroid hormones. We have previously established that apoD is a secreted protein from cultured mouse astrocytes and that treatment with the oxysterol 25-hydroxycholesterol markedly stimulates apoD release. Here, we have investigated expression and cellular processing of apoD in the Niemann-Pick type C (NPC) mouse, an animal model of human NPC, which is a genetic disorder affecting cellular cholesterol transport. NPC is phenotypically characterized by symptoms of chronic progressive neurodegeneration. ApoD gene expression was up-regulated in cultured NPC astrocytes and in NPC brain. ApoD protein levels were also increased in NPC brain with up to 30-fold higher apoD content in the NPC cerebellum compared with control mice. Subcellular fractionation of NPC brain homogenates revealed that most of the apoD was associated with the myelin fraction. ApoD was found to be a secreted protein from cultured normal astrocytes and treatment with the oxysterol, 25-hydroxycholesterol, markedly stimulated apoD release (by five- to 10-fold). By contrast, secretion of apoD from NPC astrocytes was markedly reduced and could not be stimulated by oxysterol treatment. Secretion of apoE, another apolipoprotein normally produced by astrocytes, was similar in NPC and control cells. Furthermore, apoE secretion was not potentiated by oxysterol treatment in either cell type. Plasma levels of apoD were sixfold higher in NPC, whereas hepatic levels were substantially reduced compared with controls, possibly reflecting reduced hepatic clearance of the circulating protein. These results reveal hitherto unrecognized defects in apoD metabolism in NPC that appear to be linked to the known defects in cholesterol homeostasis in this disorder.     

New Aminoacyl-tRNA Synthetase-like Protein in Insecta with an Essential Mitochondrial Function

Aminoacyl-tRNA synthetases (ARS) are modular enzymes that aminoacylate transfer RNAs (tRNA) for their use by the ribosome during protein synthesis. ARS are essential and universal components of the genetic code that were almost completely established before the appearance of the last common ancestor of all living species. This long evolutionary history explains the growing number of functions being discovered for ARS, and for ARS homologues, beyond their canonical role in gene translation. Here we present a previously uncharacterized paralogue of seryl-tRNA synthetase named SLIMP (seryl-tRNA synthetase-like insect mitochondrial protein). SLIMP is the result of a duplication of a mitochondrial seryl-tRNA synthetase (SRS) gene that took place in early metazoans and was fixed in Insecta. Here we show that SLIMP is localized in the mitochondria, where it carries out an essential function that is unrelated to the aminoacylation of tRNA. The knockdown of SLIMP by RNA interference (RNAi) causes a decrease in respiration capacity and an increase in mitochondrial mass in the form of aberrant mitochondria.     

Cholesterol as a factor regulating intracellular localization of annexin A6 in Niemann-Pick type C human skin fibroblasts

Lysosome-like storage organelles (LSOs) play a crucial role in excessive accumulation of cholesterol in the Niemann-Pick type C (NPC) disease characterized by altered vesicular traffic of lipids. Annexin A6 (AnxA6) is mainly present in cytosol but upon elevation of [Ca²⁺]_in binds to membranes. In addition, a pH or cholesterol-dependent mechanism of AnxA6 interaction with membranes was described. We found a several fold enrichment of AnxA6 in LSO compartment in fibroblasts isolated from NPC patients in comparison with fibroblasts from healthy individuals. We observed that AnxA6 relocates from cytosol to LSOs in a cholesterol-dependent manner. Cholesterol depletion caused reduction in the binding of AnxA6. Moreover, we found that in NPC cells AnxA6 translocates to the perinuclear region containing late endosomes (LE) loaded with cholesterol. We conclude that AnxA6 may participate in formation of cholesterol-rich platforms on LE and therefore may contribute to the pathology of the NPC disease.     

Proteasomes Associated with the Blm10 Activator Protein Antagonize Mitochondrial Fission through Degradation of the Fission Protein Dnm1

The conserved Blm10/PA200 activators bind to the proteasome core particle gate and facilitate turnover of peptides and unfolded proteins in vitro. We report here that Blm10 is required for the maintenance of functional mitochondria. BLM10 expression is induced 25-fold upon a switch from fermentation to oxidative metabolism. In the absence of BLM10, Saccharomyces cerevisiae cells exhibit a temperature-sensitive growth defect under oxidative growth conditions and produce colonies with dysfunctional mitochondria at high frequency. Loss of BLM10 leads to reduced respiratory capacity, increased mitochondrial oxidative damage, and reduced viability in the presence of oxidative stress or death stimuli. In the absence of BLM10, increased fragmentation of the mitochondrial network under oxidative stress is observed indicative of elevated activity of the mitochondrial fission machinery. The degradation of Dnm1, the main factor mediating mitochondrial fission, is impaired in the absence of BLM10 in vitro and in vivo. These data suggest that the mitochondrial functional and morphological changes observed are related to elevated Dnm1 levels. This hypothesis is supported by the finding that cells that constitutively overexpress DNM1 display the same mitochondrial defects as blm10Δ cells. The data are consistent with a model in which Blm10 proteasome-mediated turnover of Dnm1 is required for the maintenance of mitochondrial function and provides cytoprotection under conditions that induce increased mitochondrial damage and programmed cell death.     

Exosome Secretion Ameliorates Lysosomal Storage of Cholesterol in Niemann-Pick Type C Disease

Niemann-Pick type C1 disease is an autosomal-recessive lysosomal storage disorder. Loss of function of the npc1 gene leads to abnormal accumulation of free cholesterol and sphingolipids within the late endosomal and lysosomal compartments resulting in progressive neurodegeneration and dysmyelination. Here, we show that oligodendroglial cells secrete cholesterol by exosomes when challenged with cholesterol or U18666A, which induces late endosomal cholesterol accumulation. Up-regulation of exosomal cholesterol release was also observed after siRNA-mediated knockdown of NPC1 and in fibroblasts derived from NPC1 patients and could be reversed by expression of wild-type NPC1. We provide evidence that exosomal cholesterol secretion depends on the presence of flotillin. Our findings indicate that exosomal release of cholesterol may serve as a cellular mechanism to partially bypass the traffic block that results in the toxic lysosomal cholesterol accumulation in Niemann-Pick type C1 disease. Furthermore, we suggest that secretion of cholesterol by exosomes contributes to maintain cellular cholesterol homeostasis.     

Niemann-Pick C1 Heterogeneity of Bat Cells Controls Filovirus Tropism

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Metabolomic Profiling Reveals Biochemical Pathways and Biomarkers Associated with Pathogenesis in Cystic Fibrosis Cells

Cystic fibrosis (CF) is a life-shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To gain an understanding of the epithelial dysfunction associated with CF mutations and discover biomarkers for therapeutics development, untargeted metabolomic analysis was performed on primary human airway epithelial cell cultures from three separate cohorts of CF patients and non-CF subjects. Statistical analysis revealed a set of reproducible and significant metabolic differences between the CF and non-CF cells. Aside from changes that were consistent with known CF effects, such as diminished cellular regulation against oxidative stress and osmotic stress, new observations on the cellular metabolism in the disease were generated. In the CF cells, the levels of various purine nucleotides, which may function to regulate cellular responses via purinergic signaling, were significantly decreased. Furthermore, CF cells exhibited reduced glucose metabolism in glycolysis, pentose phosphate pathway, and sorbitol pathway, which may further exacerbate oxidative stress and limit the epithelial cell response to environmental pressure. Taken together, these findings reveal novel metabolic abnormalities associated with the CF pathological process and identify a panel of potential biomarkers for therapeutic development using this model system.     

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm²) or H₂O₂ (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1^−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity.     

Cholesterol accumulates in cell bodies,but is decreased in distal axons,of Niemann-Pick C1-deficient neurons

Niemann-Pick type-C (NPC) disease is characterized by a progressive loss of neurons and an accumulation of unesterified cholesterol within the endocytic pathway. Unlike other tissues, however, NPC1-deficient brains do not accumulate cholesterol but whether or not NPC1-deficient neurons accumulate cholesterol is not clear. Therefore, as most studies on cholesterol homeostasis in NPC1-deficient cells have been performed in fibroblasts we have investigated cholesterol homeostasis in cultured murine sympathetic neurons lacking functional NPC1. These neurons did not display obvious abnormalities in growth or morphology and appeared to respond normally to nerve growth factor. Filipin staining revealed numerous cholesterol-filled endosomes/lysosomes in NPC1-deficient neurons and the mass of cholesterol in cell bodies was greater than in wild-type neurons. Surprisingly, however, the cholesterol content of NPC1-deficient and wild-type neurons as a whole was the same. This apparent paradox was resolved when the cholesterol content of NPC1-deficient distal axons was found to be less than of wild-type axons. Cholesterol sequestration in cell bodies did not depend on exogenously supplied cholesterol since the cholesterol accumulated before birth and did not disperse when neurons were cultured without exogenous cholesterol. The altered cholesterol distribution between cell bodies and axons suggests that transport of cholesterol, particularly that synthesized endogenously, from cell bodies to distal axons is impaired in NPC1-deficient neurons.     

Endoplasmic Reticulum-associated Degradation of Niemann-Pick C1: EVIDENCE FOR THE ROLE OF HEAT SHOCK PROTEINS AND IDENTIFICATION OF LYSINE RESIDUES THAT ACCEPT UBIQUITIN*

Most cases with Niemann-Pick disease type C carry mutations in NPC1. Some of the mutations, including the most frequent I1061T, give rise to unstable proteins selected for endoplasmic reticulum-associated degradation. The purpose of the current study was to shed mechanistic insights into the degradation process. A proteasome inhibitor MG132 prolonged the life span of the wild-type NPC1 expressed in COS cells. The expressed protein associated with multiple chaperones including heat shock protein 90 (Hsp90), Hsp70, heat shock cognate protein 70 (Hsc70), and calnexin. Accordingly, expression of an E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) enhanced MG132-induced accumulation of ubiquitylated NPC1. Co-expression and RNAi knockdown experiments in HEK cells indicated that Hsp70/Hsp90 stabilized NPC1, whereas Hsc70 destabilized it. In human fibroblasts carrying the I1061T mutation, adenovirus-mediated expression of Hsp70 or treatment with an HSP-inducer geranylgeranylacetone (GGA) increased the level of the mutant protein. In GGA-treated cells, the rescued protein was localized in the late endosome and ameliorated cholesterol accumulation. MALDI-TOF mass spectrometry revealed three lysine residues at amino acids 318, 792, and 1180 as potential ubiquitin-conjugation sites. Substitutions of the three residues with alanine yielded a mutant protein with a steady-state level more than three times higher than that of the wild-type. Introduction of the same substitutions to the I1061T mutant resulted in an increase in its protein level and functional restoration. These findings indicated the role of HSPs in quality control of NPC1 and revealed the role of three lysine residues as ubiquitin-conjugation sites.     

Role of Glucose Metabolism and ATP in Maintaining PINK1 Levels during Parkin-mediated Mitochondrial Damage Responses

Mutations in several genes, including PINK1 and Parkin, are known to cause autosomal recessive cases of Parkinson disease in humans. These genes operate in the same pathway and play a crucial role in mitochondrial dynamics and maintenance. PINK1 is required to recruit Parkin to mitochondria and initiate mitophagy upon mitochondrial depolarization. In this study, we show that PINK1-dependent Parkin mitochondrial recruitment in response to global mitochondrial damage by carbonyl cyanide m-chlorophenylhydrazine (CCCP) requires active glucose metabolism. Parkin accumulation on mitochondria and subsequent Parkin-dependent mitophagy is abrogated in glucose-free medium or in the presence of 2-deoxy-d-glucose upon CCCP treatment. The defects in Parkin recruitment correlate with intracellular ATP levels and can be attributed to suppression of PINK1 up-regulation in response to mitochondria depolarization. Low levels of ATP appear to prevent PINK1 translation instead of affecting PINK1 mRNA expression or reducing its stability. Consistent with a requirement of ATP for elevated PINK1 levels and Parkin mitochondrial recruitment, local or individual mitochondrial damage via photoirradiation does not affect Parkin recruitment to damaged mitochondria as long as a pool of functional mitochondria is present in the photoirradiated cells even in glucose-free or 2-deoxy-d-glucose-treated conditions. Thus, our data identify ATP as a key regulator for Parkin mitochondrial translocation and sustaining elevated PINK1 levels during mitophagy. PINK1 functions as an AND gate and a metabolic sensor coupling biogenetics of cells and stress signals to mitochondria dynamics.     

Superoxide Triggers an Acid Burst in Saccharomyces cerevisiae to Condition the Environment of Glucose-starved Cells

Although yeast cells grown in abundant glucose tend to acidify their extracellular environment, they raise the pH of the environment when starved for glucose or when grown strictly with non-fermentable carbon sources. Following prolonged periods in this alkaline phase, Saccharomyces cerevisiae cells will switch to producing acid. The mechanisms and rationale for this “acid burst” were unknown. Herein we provide strong evidence for the role of mitochondrial superoxide in initiating the acid burst. Yeast mutants lacking the mitochondrial matrix superoxide dismutase (SOD2) enzyme, but not the cytosolic Cu,Zn-SOD1 enzyme, exhibited marked acceleration in production of acid on non-fermentable carbon sources. Acid production is also dramatically enhanced by the superoxide-producing agent, paraquat. Conversely, the acid burst is eliminated by boosting cellular levels of Mn-antioxidant mimics of SOD. We demonstrate that the acid burst is dependent on the mitochondrial aldehyde dehydrogenase Ald4p. Our data are consistent with a model in which mitochondrial superoxide damage to Fe-S enzymes in the tricarboxylic acid (TCA) cycle leads to acetate buildup by Ald4p. The resultant expulsion of acetate into the extracellular environment can provide a new carbon source to glucose-starved cells and enhance growth of yeast. By triggering production of organic acids, mitochondrial superoxide has the potential to promote cell population growth under nutrient depravation stress.     

The Role of Mitochondrial dNTP Levels in Cells with Reduced TK2 Activity

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.     

Stress-triggered Activation of the Metalloprotease Oma1 Involves Its C-terminal Region and Is Important for Mitochondrial Stress Protection in Yeast

Functional integrity of mitochondria is critical for optimal cellular physiology. A suite of conserved mitochondrial proteases known as intramitochondrial quality control represents one of the mechanisms assuring normal mitochondrial function. We previously demonstrated that ATP-independent metalloprotease Oma1 mediates degradation of hypohemylated Cox1 subunit of cytochrome c oxidase and is active in cytochrome c oxidase-deficient mitochondria. Here we show that Oma1 is important for adaptive responses to various homeostatic insults and preservation of normal mitochondrial function under damage-eliciting conditions. Changes in membrane potential, oxidative stress, or chronic hyperpolarization lead to increased Oma1-mediated proteolysis. The stress-triggered induction of Oma1 proteolytic activity appears to be associated with conformational changes within the Oma1 homo-oligomeric complex, and these alterations likely involve C-terminal residues of the protease. Substitutions in the conserved C-terminal region of Oma1 impair its ability to form a labile proteolytically active complex in response to stress stimuli. We demonstrate that Oma1 genetically interacts with other inner membrane-bound quality control proteases. These findings indicate that yeast Oma1 is an important player in IM protein homeostasis and integrity by acting in concert with other intramitochondrial quality control components.     

Niemann-Pick Type C1 deficiency in microglia does not cause neuron death in vitro

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of cholesterol and other lipids in late endosomes/lysosomes and leads to progressive neurodegeneration and premature death. The mechanism by which lipid accumulation causes neurodegeneration remains unclear. Inappropriate activation of microglia, the resident immune cells of the central nervous system, has been implicated in several neurodegenerative disorders including NPC disease. Immunohistochemical analysis demonstrates that NPC1 deficiency in mouse brains alters microglial morphology and increases the number of microglia. In primary cultures of microglia from Npc1^−/− mice cholesterol is sequestered intracellularly, as occurs in other NPC-deficient cells. Activated microglia secrete potentially neurotoxic molecules such as tumor necrosis factor-α (TNFα). However, NPC1 deficiency in isolated microglia did not increase TNFα mRNA or TNFα secretion in vitro. In addition, qPCR analysis shows that expression of pro-inflammatory and oxidative stress genes is the same in Npc1^+/+ and Npc1^−/− microglia, whereas the mRNA encoding the anti-inflammatory cytokine, interleukin-10 in Npc1^−/− microglia is ~ 60% lower than in Npc1^+/+ microglia. The survival of cultured neurons was not impaired by NPC1 deficiency, nor was death of Npc1^−/− and Npc1^+/+ neurons in microglia-neuron co-cultures increased by NPC1 deficiency in microglia. However, a high concentration of Npc1^−/− microglia appeared to promote neuron survival. Thus, although microglia exhibit an active morphology in NPC1-deficient brains, lack of NPC1 in microglia does not promote neuron death in vitro in microglia-neuron co-cultures, supporting the view that microglial NPC1 deficiency is not the primary cause of neuron death in NPC disease.     

Induction of a Tumor-associated Activating Mutation in Protein Tyrosine Phosphatase Ptpn11 (Shp2) Enhances Mitochondrial Metabolism,Leading to Oxidative Stress and Senescence

Activating mutations in Ptpn11 (Shp2), a protein tyrosine phosphatase involved in diverse cell signaling pathways, are associated with pediatric leukemias and solid tumors. However, the pathogenic effects of these mutations have not been fully characterized. Here, we report that induction of the Ptpn11^E76K/+ mutation, the most common and active Ptpn11 mutation found in leukemias and solid tumors, in primary mouse embryonic fibroblasts resulted in proliferative arrest and premature senescence. As a result, apoptosis was markedly increased. These cellular responses were accompanied and mediated by up-regulation of p53 and p21. Moreover, intracellular levels of reactive oxygen species (ROS), byproducts of mitochondrial oxidative phosphorylation, were elevated in Ptpn11^E76K/+ cells. Since Shp2 is also distributed to the mitochondria (in addition to the cytosol), the impact of the Ptpn11^E76K/+ mutation on mitochondrial function was analyzed. These analyses revealed that oxygen consumption of Ptpn11^E76K/+ cells and the respiratory function of Ptpn11^E76K/+ mitochondria were significantly increased. Furthermore, we found that phosphorylation of mitochondrial Stat3, one of the substrates of Shp2 phosphatase, was greatly decreased in the mutant cells with the activating mutation Ptpn11^E76K/+. This study provides novel insights into the initial effects of tumor-associated Ptpn11 mutations.     

The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b₅ reductase (CYB5R) and cytochrome b₅ (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.     

Metabolic Analysis of Wild-type Escherichia coli and a Pyruvate Dehydrogenase Complex (PDHC)-deficient Derivative Reveals the Role of PDHC in the Fermentative Metabolism of Glucose

Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of ¹³C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional ¹³C,¹H NMR spectroscopy. Metabolite balancing, biosynthetic ¹³C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO₂ and the calculated CO₂ evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO₂ for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO₂, thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO₂ and H₂) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO₂ generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth.