Characterization of the interaction between Aβ 1–42 and glyceraldehyde phosphodehydrogenase

Advances in the understanding of AD pathogenesis have recently provided strong support for a modified Aβ protein cascade hypothesis, stating that several different Aβ assemblies contribute to the triggering of a complex pathological cascade leading to neurodegeneration. Both in vitro and in vivo, Aβ rapidly forms fibrils (fAβ), which are able to interact with various molecular partners, including proteins, lipids and proteoglycans. In a previous study aimed to identify some of these molecular partners of fAβ, we demonstrated that the GAPDH was specifically coprecipitated with fAβ. The aim of this study was to characterize this interaction. First, it was shown by TEM that synthetic GAPDH directly binds fAβ 1–42. Then rat synaptosomal proteins were purified and incubated with different forms of Aβ in various conditions, and the presence of GAPDH among the proteins coprecipitated with Aβ was studied by western blotting. Results showed that the interaction between GAPDH and fAβ 1–42 is nonionic, as is not impaired by increasing salt concentrations. GAPDH is coprecipitated not only by fAβ, but also by nonfibrillar forms of Aβ 1–42. The 41–42 Aβ sequence seems to be important in the interaction of GAPDH and Aβ, as more GAPDH was coprecipitated with fAβ 1–42 than with fAβ 1–40. GAPDH extracted from various subcellular fractions including mitochondria, was shown to interact with fAβ. Our data demonstrate a direct interaction between Aβ and GAPDH and support the possibility that this interaction has an important pathogenic role in AD. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

A study of binding-solubilization of some glycolytic enzymes in striated muscle in situ

The solubilization of lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and alpha-glycerophosphate dehydrogenase (GAPDH) was studied in pressed muscle as a function of ionic strength and NADH concentration. The results indicate that these factors affect the binding-solubilization of LDH and GAPDH in a similar way to their effect in dilute homogenized tissue. Alpha-glycerophosphate dehydrogenase was included as a typical soluble enzyme, since we have been unable to demonstrate its binding to subcellular fractions under any conditions. As with homogenized tissue, LDH was less susceptible to solubilization by ionic strength than GAPDH. It was demonstrated that LDH isozymes richer in heart-type subunits were more easily removed from muscle by centrifugation-imbibition than isozymes richer in the muscle-type subunits. This was interpreted as indicating that binding of the enzyme to subcellular structures was a major factor in the restricted removal of these enzymes from muscle, since only the muscle-type subunit is capable of binding to subcellular particles. It was further demonstrated that LDH could be taken up into muscle tissue, depleted in the enzyme, against an apparent concentration gradient. This was also interpreted as binding of the enzyme to the particulate structure of the muscle. Furthermore, this uptake of LDH occurred under conditions of ionic strength (0.25) and pH (7.5) that would prevent binding of the enzyme to the particulate fraction of a dilute suspension of homogenized muscle tissue. Thus, physiological conditions of pH and ionic strength do not necessarily induce solubilization of chicken breast muscle LDH in situ. Data obtained with dilute tissue homogenates, therefore, may not necessarily be easily and safely extrapolated to conditions in situ.     

Subcellular Distribution of Glyceraldehyde-3-Phosphate Dehydrogenase in Cerebellar Granule Cells Undergoing Cytosine Arabinoside-Induced Apoptosis

Abstract: We have previously shown that cytosine arabinoside (AraC)-induced apoptosis of cerebellar granule cells (CGCs) results in an increase of a 38-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). Antisense oligonucleotides to GAPDH mRNA afford acutely plated CGCs significant protection against AraC-induced apoptosis. We used differential centrifugation to examine which subcellular components are affected. Treated and untreated cells were sonicated in 0.32 M sucrose and sequentially centrifuged at 1,000, 20,000, and 200,000 g , to obtain crude nuclear, mitochondrial, microsomal, and cytosolic fractions. Western blotting showed that the levels of GAPDH protein were markedly increased in the 1,000- and 20,000- g pellets. The levels in the cytosolic supernatant were decreased dramatically by AraC in acutely plated CGCs but not in cells 24 h after plating. It is noteworthy that although GAPDH protein in the pellet fractions increased, the dehydrogenase activity of GAPDH decreased. Two other dehydrogenases, lactate dehydrogenase (EC 1.1.1.27) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49), were not similarly affected, suggesting that the effect was GAPDH specific. These observations suggest that GAPDH levels change in specific organelles during apoptosis for reasons that are separate from its function as a glycolytic enzyme. The accumulation of GAPDH protein in specific subcellular loci may play a role in neuronal apoptosis.     

In vivo incorporation of [U-14C]glycerol and [2-3H]glycerol into phospholipids of brain subcellular fractions in normal and malnourished animals

A double label design was used to study the in vivo incorporation of [U-¹⁴C] and [2-³H]glycerol into total and individual phospholipids of various brain subcellular fractions isolated from 20-day old normal and undernourished rats. In control animals, synthesis of glycerophospholipids of microsomes, mitochondria and nerve endings seems to occur through the glycerol-3-phosphate (G-3-P) pathway while a large part of the synthesis of myelin glycerophospholipids appears to proceed through the dihydroxyacetone phosphate (DHAP) pathway. In starved animals, on the other hand the incorporation of phospholipid precursors through the DHAP pathway was found to be lower than in controls while synthesis of phospholipids in the other subcellular fractions was unaffected.The possible relationship between the synthesis of glycerophospholipids and especially plasmalogens of the myelin membrane and microperoxisomes of oligodendroglial cells, where the enzymes of the DHAP pathway are located, is discussed.     

Reduction of glyceraldehyde-3-phosphate dehydrogenase activity in Alzheimer's disease and in Huntington's disease fibroblasts

New functions have been identified for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) including its role in neurodegenerative disease and in apoptosis. GAPDH binds specifically to proteins implicated in the pathogenesis of a variety of neurodegenerative disorders including the beta-amyloid precursor protein and the huntingtin protein. However, the pathophysiological significance of such interactions is unknown. In accordance with published data, our initial results indicated there was no measurable difference in GAPDH glycolytic activity in crude whole-cell sonicates of Alzheimer's and Huntington's disease fibroblasts. However, subcellular-specific GAPDH-protein interactions resulting in diminution of GAPDH glycolytic activity may be disrupted or masked in whole-cell preparations. For that reason, we examined GAPDH glycolytic activity as well as GAPDH-protein distribution as a function of its subcellular localization in 12 separate cell strains. We now report evidence of an impairment of GAPDH glycolytic function in Alzheimer's and Huntington's disease subcellular fractions despite unchanged gene expression. In the postnuclear fraction, GAPDH was 27% less glycolytically active in Alzheimer's cells as compared with age-matched controls. In the nuclear fraction, deficits of 27% and 33% in GAPDH function were observed in Alzheimer's and Huntington's disease, respectively. This evidence supports a functional role for GAPDH in neurodegenerative diseases. The possibility is considered that GAPDH:neuronal protein interaction may affect its functional diversity including energy production and as well as its role in apoptosis.     

Glyceraldehyde-3-phosphate dehydrogenase,a glycolytic enzyme present in the periplasm of Aeromonas hydrophila

This is the first report describing the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a protein associated with the cell envelope of a gram-negative bacterium (Aeromonas hydrophila). Dose-dependent GAPDH activity was detected in whole bacterial cells from exponentially growing cultures, indicating that an active form of GAPDH is located outside the plasma membrane. This activity represents roughly 10–20% of total cell activity, and it is not reduced by pretreatment of the cells with trypsin. Assays with soluble GAPDH indicate that the activity measured in intact cells does not originate by rebinding to intact cells of cytosolic enzyme released following cell lysis. GAPDH activity levels detected in intact cells varied during the growth phase. The relationship between GAPDH activity and cell culture density was not linear, showing this activity as a major peak in the late-logarithmic phase (A₆₀₀ = 1.1–1.3), and a decrease when cells entered the stationary phase. The late exponential growing cells showed a GAPDH activity 3 to 4-fold higher than early growing or stationary cells. No activity was detected in culture supernatants. Enzymatic and Western-immunoblotting analysis of subcellular fractions (cytosol, whole and outer membranes, and periplasm) showed that GAPDH is located in the cytosol, as expected, and also in the periplasm. These results place the periplasmic GAPDH of A. hydrophila into the family of multifunctional microbial cell wall-associated GAPDHs which retain their catalytic activity. This revised version was published online in June 2006 with corrections to the Cover Date.     

GAPDH is not regulated in human glioblastoma under hypoxic conditions

Background

Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

Results

We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.

Conclusion

GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.     

Studies on Tetrahymena membranes:: Temperature-induced alterations in fatty acid composition of various membrane fractions in Tetrahymena pyriformis and its effect on membrane fluidity as inferred by spin-label study

The fatty acid distribution pattern of lipids extracted from different subcellular components of Tetrahymena pyriformis was found to be significantly different from one type of membrane to another.The growth-temperature shift caused alterations in fatty acid composition. The ratio of palmitoleic to palmitic acid, especially, showed a sharp linear decline with increase of temperature in all of the membrane fractions.The spin labels were rapidly incorporated into Tetrahymena membranes. The order parameter of 5-nitroxide stearate spin label incorporated into various membrane fractions was found to be different for the different membrane fractions, suggesting the following order of the fluidity; microsomes > pellicles > cilia.The fluidity of the surface membranes, cilia and pellicles isolated from Tetrahymena cells grown at 15°C was noticeably higher than that of the membranes from cells grown at 34°C but was not so different with microsomal fractions.The motion of the spin label in the pellicular membrane was more restricted than in its extracted lipids, thus indicating the assumption that in Tetrahymena membranes the proteins influence the fluidity.It was also suggested that a sterol-like triterpenoid compound, tetrahymanol, which is principally localized in the surface membranes, would be involved in the membrane fluidity.     

Interaction of Cd and Zn during uptake and loss in the polychaete Capitella capitata: Whole body and subcellular perspectives

The interaction between Cd and Zn in aquatic organisms is known to be highly variable. The purpose of this study was to use a subcellular compartmentalization approach to examine Cd and Zn interactions in the deposit-feeding polychaete Capitella capitata (sp. I). Laboratory-reared C. capitata were co-exposed to Cd (background or 50 μg Cd l^− 1) and Zn (background or 86 μg Zn l^− 1) with ¹⁰⁹Cd and ⁶⁵Zn as radiotracers for 1 week. After the 1-week uptake period, subsets of worms were allowed to depurate accumulated metals for an additional 1 week. Worms from both phases (uptake and loss) were then subjected to subcellular fractionation to determine the compartmentalization of metals as metal-sensitive fractions [MSF — organelles and heat-denaturable proteins (HDP)] and biologically detoxified metals [BDM — heat-stable proteins (HSP) and metal-rich granules (MRG)]. Uptake and loss of Cd and Zn in C. capitata at the whole body level were similar at bkgd-Cd/bkgd-Zn, with worms depurating the majority of accumulated metal (∼ 75% Cd and ∼ 64% Zn). When exposure of Zn or Cd was increased (bkgd-Cd/86-Zn; bkgd-Zn/50-Cd), uptake of background levels of Cd or Zn, respectively, was suppressed by ∼ 50%. These accumulated metals, however, were retained during the loss phase resulting in ∼ 40-50% greater Cd and Zn whole body tissue burdens than those of bkgd-Cd/bkgd-Zn worms. Beyond exhibiting similar patterns of uptake and loss at the whole body level, Cd and Zn behaved similarly at the subcellular level. Under background levels (bkgd-Cd/bkgd-Zn), after uptake, worms partitioned a majority of Cd (∼ 65%) and Zn (∼ 55%) to the HSP and organelles fractions. The HDP and MRG fractions contained less than ∼ 6% of both metals. Following depuration, at bkgd-Cd/bkgd-Zn, Cd and Zn were lost from all subcellular fractions; loss from HSP was the greatest contributor to whole body loss. When exposed to elevated concentrations of Zn or Cd, the suppression in uptake of bkgd-Cd or bkgd-Zn observed in whole body uptake was largely due to suppressions in the storage of Cd and Zn to HSP. These results suggest that Cd-Zn interactions reduce partitioning of both Cd and Zn to HSP, indicating that metal-binding proteins such as metallothioneins play a key role in these interactions.     

Generation of transgenic Arabidopsis plants expressing mcherry-fused organelle marker proteins

In eukaryotic cells, a major proportion of the cellular proteins localize to various subcellular organelles where they are involved in organelle-specific cellular processes. Thus, the localization of a particular protein in the cell is an important part of understanding the physiological role of the protein in the cell. Various approaches such as subcellular fractionation, immunolocalization and live imaging have been used to define the localization of organellar proteins. Of these various approaches, the most powerful one is the live imaging because it can show in vivo dynamics of protein localization depending on cellular and environmental conditions without disturbing cellular structures. However, the live imaging requires the ability to detect the organelles in live cells. In this study, we report generation of a new set of transgenic Arabidopsis plants using various organelle marker proteins fused to a fluorescence protein, monomeric Cherry (mCherry). All these markers representing different subcellular organelles such as chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER) and lytic vacuole showed clear and specific signals regardless of the cell types and tissues. These marker lines can be used to determine localization of organellar proteins by colocalization and also to study the dynamics of organelles under various developmental and environmental conditions.     

New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein examined for its pivotal role in energy production. It was also used as a model protein for analysis of protein structure and enzyme mechanisms. The GAPDH gene was utilized as a prototype for studies of genetic organization, expression and regulation. However, recent evidence demonstrates that mammalian GAPDH displays a number of diverse activities unrelated to its glycolytic function. These include its role in membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and DNA repair. These new activities may be related to the subcellular localization and oligomeric structure of GAPDH in vivo. Furthermore, other investigations suggest that GAPDH is involved in apoptosis, age-related neurodegenerative disease, prostate cancer and viral pathogenesis. Intriguingly, GAPDH is also a unique target of nitric oxide. This review discusses the functional diversity of GAPDH in relation to its protein structure. The mechanisms through which mammalian cells may utilize GAPDH amino acid sequences to provide these new functions and to determine its intracellular localization are considered. The interrelationship between new GAPDH activities and its role in cell pathologies is addressed.     

A Dimer Interface Mutation in Glyceraldehyde-3-Phosphate Dehydrogenase Regulates Its Binding to AU-rich RNA

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3′-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD⁺ binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.     

Aging of human glyceraldehyde-3-phosphate dehydrogenase is dependent on its subcellular localization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), long considered a traditional glycolytic protein, displays multiple activities independent of its role in energy generation. This functional diversity is dependent on its membrane, cytoplasmic or nuclear localization. GAPDH is encoded by one active gene and is synthesized as a single 37 kDa protein without alternate splicing. Accordingly, the identical protein would be present in each subcellular fraction. The accumulation of post-translational errors in protein structure as a function of oxidative stress is thought to provide a basic molecular mechanism for the aging process. Thus, during aging, the GAPDH protein should contain the identical degree of oxidative sequence alteration irrespective of its distribution. This would result in equivalent effects on GAPDH activity. However, conformational differences in GAPDH structure due to its subcellular protein, nucleic acid or membrane interactions could affect its degree of modification thereby selectively affecting its function. For that reason, we examined the subcellular expression and intracellular activity of GAPDH as a function of human aging. Subcellular GAPDH expression was quantitated by immunoblot analysis in fetal and senior human cells (postnuclear, nuclear, perinuclear). GAPDH activity was determined by in vitro assay. We now report that the aging of human GAPDH was subcellular dependent. Reductions of nuclear and postnuclear GAPDH activity in senior cells were twofold lower than that observed for the perinuclear protein. In contrast, the subcellular expression of the GAPDH protein was age-independent. These results suggest the possibility that subcellular interactions may mitigate oxidative stress-induced GAPDH modification in human aging. Such selective effects on GAPDH could affect its functional diversity.     

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD⁺/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.     

Plasmodium: high-voltage electron microscopy

Stereoscopy of thick sections (~1.0 μm) of biological material, Plasmodium gallinaceum and Plasmodium berghei, was investigated with a high-voltage electron microscope using an accelerating voltage of 650 kv. Not only could structural details of the malarial parasites be observed in the thick sections, but stereoscopic examination also revealed the physical relationships of the various subcellular organelles in the parasites. This study indicates that the high-voltage electron microscope is a useful tool for structural analysis of malarial parasites.     

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.     

Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart

Mitochondrial proteins have been shown to be common targets of S-nitrosylation (SNO), but the existence of a mitochondrial source of nitric oxide remains controversial. SNO is a nitric oxide-dependent thiol modification that can regulate protein function. Interestingly, trans-S-nitrosylation represents a potential pathway for the import of SNO into the mitochondria. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown to act as a nuclear trans-S-nitrosylase, has also been shown to enter mitochondria. However, the function of GAPDH in the mitochondria remains unknown. Therefore, we propose the hypothesis that S-nitrosylated GAPDH (SNO-GAPDH) interacts with mitochondrial proteins as a trans-S-nitrosylase. In accordance with this hypothesis, SNO-GAPDH should be detected in mitochondrial fractions, interact with mitochondrial proteins, and increase mitochondrial SNO levels. Our results demonstrate a four-fold increase in GAPDH levels in the mitochondrial fraction of mouse hearts subjected to ischemic preconditioning, which increases SNO-GAPDH levels. Co-immunoprecipitation studies performed in mouse hearts perfused with the S-nitrosylating agent S-nitrosoglutathione (GSNO), suggest that SNO promotes the interaction of GAPDH with mitochondrial protein targets. The addition of purified SNO-GAPDH to isolated mouse heart mitochondria demonstrated the ability of SNO-GAPDH to enter the mitochondrial matrix, and to increase SNO for many mitochondrial proteins. Further, the overexpression of GAPDH in HepG2 cells increased SNO for a number of different mitochondrial proteins, including heat shock protein 60, voltage-dependent anion channel 1, and acetyl-CoA acetyltransferase, thus supporting the role of GAPDH as a potential mitochondrial trans-S-nitrosylase. In further support of this hypothesis, many of the mitochondrial SNO proteins identified with GAPDH overexpression were no longer detected with GAPDH knock-down or mutation. Therefore, our results suggest that SNO-GAPDH can act as a mitochondrial trans-S-nitrosylase, thereby conferring the transfer of SNO from the cytosol to the mitochondria.