Penetration of the mitochondrial membrane by glutamate and aspartate

doi:10.1016/0006-291X(67)90556-6

Biochemical and Biophysical Research Communications

Volume 29, Issue 1, 11 October 1967, Pages 148-152

https://doi.org/10.1016/0006-291X(67)90556-6 Get rights and content

Abstract

Evidence has been presented for the existence of systems located in mitochondrial membranes for the transport of phosphate (Chappell & Crofts, 1966), dicarboxylic acids (Chappell & Haarhoff, 1967; Robinson & Chappell, 1967), citrate, isocitrate and cis-aconitate (Chappell, 1964, Chappell, 1966; Chappell & Haarhoff, 1967; Chappell, Henderson, McGivan & Robinson, 1967) and for oxoglutarate (Meijer & Tager, 1966; Chappell et al., 1967). In this paper results are presented which reveal the probable existence of two more carrier-systems, one for L-glutamate and one for L-aspartate. The latter carrier requires the presence of L-glutamate, or certain analogues of this amino acid, before it is able to act. The evidence for the glutamate carrier is, firstly, that the rate of reduction of intramitochondrial NAD(P) by glutamate has an optimum below pH 6, whereas with the free dehydrogenase the pH optimum is greater than 8. Secondly, certain analogues of glutamate, namely 3-hydroxyglutamate, 2-aminoadipate and threo-hydroxyaspartate inhibit competitively the reduction of intramitochondrial NAD(P) by glutamate, but have no or negligible effect on the activity of enzyme in mitochondrial extracts. The evidence for the aspartate carrier rests on the fact that aspartate is unable to react rapidly with mitochondrial aspartate aminotransferase until glutamate, or of the compounds tested hydroxyglutamate, aminoadipate or threo-hydroxyaspartate, are present.

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Citrin deficiency is inherited in an autosomal recessive manner and is caused by pathogenic variants of the SLC25A13 gene, which encodes the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), also called citrin [20,21]. Citrin is located in the mitochondrial inner membrane and is responsible for the symport of glutamate with a proton into the mitochondrial matrix and the export of aspartate from the matrix to the cytoplasm [21–26], a critical step in the urea cycle [27], gluconeogenesis [28], the malate-aspartate shuttle [29,30], and metabolic energy production. There are two human isoforms of the mitochondrial aspartate/glutamate carrier.
Citrin deficiency is a pan-ethnic and highly prevalent mitochondrial disease with three different stages: neonatal intrahepatic cholestasis (NICCD), a relatively mild adaptation stage, and type II citrullinemia in adulthood (CTLN2). The cause is the absence or dysfunction of the calcium-regulated mitochondrial aspartate/glutamate carrier 2 (AGC2/SLC25A13), also called citrin, which imports glutamate into the mitochondrial matrix and exports aspartate to the cytosol. In citrin deficiency, these missing transport steps lead to impairment of the malate-aspartate shuttle, gluconeogenesis, amino acid homeostasis, and the urea cycle. In this review, we describe the geological spread and occurrence of citrin deficiency, the metabolic consequences and use our current knowledge of the structure to predict the impact of the known pathogenic mutations on the calcium-regulatory and transport mechanism of citrin.
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Since mitochondria are responsible for the majority of ROS production in cells, a significant amount of GSH is transported into the mitochondria. Two inner mitochondrial membrane anion transporters, the dicarboxylate carrier (DIC, Slc25a10) and the 2-oxoglutarate carrier (OGC, Slc25a11) transport GSH into the mitochondria (Azzi et al., 1967; Lash, 2006; Meijer et al., 1972). Mitochondria receive 10%–15% of the total cellular GSH that equalizes the mitochondrial GSH concentration to the cytosolic GSH concentration (Griffith and Meister, 1985).
Redox balance is essential for normal brain, hence dis-coordinated oxidative reactions leading to neuronal death, including programs of regulated death, are commonly viewed as an inevitable pathogenic penalty for acute neuro-injury and neurodegenerative diseases. Ferroptosis is one of these programs triggered by dyshomeostasis of three metabolic pillars: iron, thiols, and polyunsaturated phospholipids. This review focuses on: (1) lipid peroxidation (LPO) as the major instrument of cell demise, (2) iron as its catalytic mechanism, and (3) thiols as regulators of pro-ferroptotic signals, hydroperoxy lipids. Given the central role of LPO, we discuss the engagement of selective and specific enzymatic pathways versus random free radical chemical reactions in the context of the phospholipid substrates, their biosynthesis, intracellular location, and related oxygenating machinery as participants in ferroptotic cascades. These concepts are discussed in the light of emerging neuro-therapeutic approaches controlling intracellular production of pro-ferroptotic phospholipid signals and their non-cell-autonomous spreading, leading to ferroptosis-associated necroinflammation.
The SLC25 Mitochondrial Carrier Family: Structure and Mechanism
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The aspartate/glutamate carriers AGC1 (SCL25A12) and AGC2 (SLC25A13) are examples of amino acid transporters. They import glutamate in symport with a proton and export aspartate, and they function in the malate-aspartate shuttle, gluconeogenesis, the urea cycle (AGC2-specific), and myelin synthesis (AGC1-specific) [27,28]. They are calcium regulated and have an unusual three-domain structure consisting of an N-terminal calcium-regulatory domain containing eight EF-hands, a carrier domain, and a C-terminal amphipathic helix [29].
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Glutamate is implicated in numerous metabolic and signalling functions that vary according to specific tissues. Glutamate metabolism is tightly controlled by activities of mitochondrial enzymes and transmembrane carriers, in particular glutamate dehydrogenase and mitochondrial glutamate carriers that have been identified in recent years. It is remarkable that, although glutamate-specific enzymes and transporters share similar properties in most tissues, their regulation varies greatly according to particular organs in order to achieve tissue specific functions. This is illustrated in this review when comparing glutamate handling in liver, brain, and pancreatic β-cells. We describe the main cellular glutamate pathways and their specific functions in different tissues, ultimately contributing to the control of metabolic homeostasis at the organism level.
Identification of the mitochondrial glutamate transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms
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Therefore, with both GC1 and GC2, the Vmax value of the glutamate/H+ symport is markedly stimulated by the trans-membrane pH gradient, whereas the Vmaxvalue of the glutamate/glutamate exchange is almost unaffected, as observed for carriers that catalyze a proton symport. The existence of a specific transporter for glutamate was inferred first in 1967 from studies of the reduction of mitochondrial NAD(P) by glutamate and of the swelling of liver mitochondria in ammonium glutamate (30). Since then, its main properties have been studied in intact mitochondria (15-24).
The mitochondrial carriers are a family of transport proteins in the inner membranes of mitochondria. They shuttle substrates, metabolites, and cofactors through this membrane and connect cytoplasm functions with others in the matrix. Glutamate is co-transported with H⁺ (or exchanged for OH⁻), but no protein has ever been associated with this activity. Two human expressed sequence tags encode proteins of 323 and 315 amino acids with 63% identity that are related to the aspartate-glutamate carrier, a member of the carrier family. They have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties demonstrate that the two proteins are isoforms of the glutamate/H⁺ symporter described in the past in whole mitochondria. Isoform 1 is expressed at higher levels than isoform 2 in all the tissues except in brain, where the two isoforms are expressed at comparable levels. The differences in expression levels and kinetic parameters of the two isoforms suggest that isoform 2 matches the basic requirement of all tissues especially with respect to amino acid degradation, and isoform 1 becomes operative to accommodate higher demands associated with specific metabolic functions such as ureogenesis.
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1992, Comparative Biochemistry and Physiology -- Part B: Biochemistry and
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  1. Proline accumulation by tsetse fly Glossina morsitans flight muscle mitochondria was studied in vitro by the swelling technique and direct measurement of (U-¹⁴C) proline.
- 2.
  2. Proline transport was inhibited by the uncharged liposoluble-SH reagent, N-ethylmaleimide but not by ionic reagent, mersalyl, suggesting that the -SH groups involved in the transport of proline are located in a hydrophobic part of the membrane or on the matrix side of the membrane.
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  3. The kineticy study of proline accumulation revealed saturation kinetics and a high temperature dependence. It gave a K_m of 85 μM and a V_max of 962 pmol/min/mg protein and an activation energy (E_a) of 11 kcal/mol.
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  4. Certain other amino acids (l-valine, l-alanine, l-methionine, l-phenylalanine, l-tryptophan and l-hydroxyproline) significantly stimulated proline uptake.
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  5. These observations indicate that tsetse fly Glossina morsians flight muscle mitochondria contain a proline transport mechanism.

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Penetration of the mitochondrial membrane by glutamate and aspartate

Abstract

Biochem. Biophys. Res. Comm

Biochem. J

Biochem. J