Glutamine-Derived Aspartate Biosynthesis in Cancer Cells: Role of Mitochondrial Transporters and New Therapeutic Perspectives

Aspartate has a central role in cancer cell metabolism. Aspartate cytosolic availability is crucial for protein and nucleotide biosynthesis as well as for redox homeostasis. Since tumor cells display poor aspartate uptake from the external environment, most of the cellular pool of aspartate derives from mitochondrial catabolism of glutamine. At least four transporters are involved in this metabolic pathway: the glutamine (SLC1A5_var), the aspartate/glutamate (AGC), the aspartate/phosphate (uncoupling protein 2, UCP2), and the glutamate (GC) carriers, the last three belonging to the mitochondrial carrier family (MCF). The loss of one of these transporters causes a paucity of cytosolic aspartate and an arrest of cell proliferation in many different cancer types. The aim of this review is to clarify why different cancers have varying dependencies on metabolite transporters to support cytosolic glutamine-derived aspartate availability. Dissecting the precise metabolic routes that glutamine undergoes in specific tumor types is of upmost importance as it promises to unveil the best metabolic target for therapeutic intervention.

Significance of AtMTM1 and AtMTM2 for Mitochondrial MnSOD Activation in Arabidopsis

The manganese (Mn) tracking factor for mitochondrial Mn superoxide dismutase (MnSOD) has been annotated as yMTM1 in yeast, which belongs to the mitochondrial carrier family. We confirmed that Arabidopsis AtMTM1 and AtMTM2 are functional homologs of yMYM1 as they can revive yeast MnSOD activity in yMTM1-mutant cells. Transient expression of AtMnSOD-3xFLAG in the AtMTM1 and AtMTM2-double mutant protoplasts confirmed that AtMTM1 and AtMTM2 are required for AtMnSOD activation. Our study revealed that AtMnSOD interacts with AtMTM1 and AtMTM2 in the mitochondria. The expression levels of AtMTM1, AtMTM2, and AtMnSOD respond positively to methyl viologen (MV) and metal stress. AtMTM1 and AtMTM2 are involved in Mn and Fe homeostasis, root length, and flowering time. Transient expression of chloroplast-destined AtMnSOD revealed that an evolutionarily conserved activation mechanism, like the chloroplastic-localized MnSOD in some algae, still exists in Arabidopsis chloroplasts. This study strengthens the proposition that AtMTM1 and AtMTM2 participate in the AtMnSOD activation and ion homeostasis.

Computational studies of the mitochondrial carrier family SLC25. Present status and future perspectives

The members of the mitochondrial carrier family, also known as solute carrier family 25 (SLC25), are transmembrane proteins involved in the translocation of a plethora of small molecules between the mitochondrial intermembrane space and the matrix. These transporters are characterized by three homologous domains structure and a transport mechanism that involves the transition between different conformations. Mutations in regions critical for these transporters’ function often cause several diseases, given the crucial role of these proteins in the mitochondrial homeostasis. Experimental studies can be problematic in the case of membrane proteins, in particular concerning the characterization of the structure–function relationships. For this reason, computational methods are often applied in order to develop new hypotheses or to support/explain experimental evidence. Here the computational analyses carried out on the SLC25 members are reviewed, describing the main techniques used and the outcome in terms of improved knowledge of the transport mechanism. Potential future applications on this protein family of more recent and advanced in silico methods are also suggested.

Antioxidant Synergy of Mitochondrial Phospholipase PNPLA8/iPLA2γ with Fatty Acid–Conducting SLC25 Gene Family Transporters

Patatin-like phospholipase domain-containing protein PNPLA8, also termed Ca2+-independent phospholipase A2γ (iPLA2γ), is addressed to the mitochondrial matrix (or peroxisomes), where it may manifest its unique activity to cleave phospholipid side-chains from both sn-1 and sn-2 positions, consequently releasing either saturated or unsaturated fatty acids (FAs), including oxidized FAs. Moreover, iPLA2γ is directly stimulated by H2O2 and, hence, is activated by redox signaling or oxidative stress. This redox activation permits the antioxidant synergy with mitochondrial uncoupling proteins (UCPs) or other SLC25 mitochondrial carrier family members by FA-mediated protonophoretic activity, termed mild uncoupling, that leads to diminishing of mitochondrial superoxide formation. This mechanism allows for the maintenance of the steady-state redox status of the cell. Besides the antioxidant role, we review the relations of iPLA2γ to lipid peroxidation since iPLA2γ is alternatively activated by cardiolipin hydroperoxides and hypothetically by structural alterations of lipid bilayer due to lipid peroxidation. Other iPLA2γ roles include the remodeling of mitochondrial (or peroxisomal) membranes and the generation of specific lipid second messengers. Thus, for example, during FA β-oxidation in pancreatic β-cells, H2O2-activated iPLA2γ supplies the GPR40 metabotropic FA receptor to amplify FA-stimulated insulin secretion. Cytoprotective roles of iPLA2γ in the heart and brain are also discussed.

Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes

The mitochondrial carrier family protein SLC25A3 transports both copper and phosphate in mammals yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2 and MIR1. To understand the ancestral state of copper and phosphate transport in mitochondria, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses revealed that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating an ancient gene duplication created these paralogs. To link this phylogenetic signal to protein function, we used structural modelling and site-directed mutagenesis to identify residues involved in copper and phosphate transport. Based on these analyses, we generated a L175A variant of mouse SLC25A3 that retains the ability to transport copper but not phosphate. This work highlights the utility of using an evolutionary framework to uncover amino acids involved in substrate recognition by mitochondrial carrier family proteins.

ER targeting of non-imported mitochondrial carrier proteins is dependent on the GET pathway

Deficiencies in mitochondrial import cause the toxic accumulation of non-imported mitochondrial precursor proteins. Numerous fates for non-imported mitochondrial precursors have been identified in budding yeast, including proteasomal destruction, deposition into protein aggregates, and mistargeting to other organelles. Amongst organelles, the ER has emerged as a key destination for a subset of non-imported mitochondrial proteins. However, how ER targeting of various types of mitochondrial proteins is achieved remains incompletely understood. Here, we show that the ER delivery of endogenous mitochondrial transmembrane proteins, especially those belonging to the SLC25A mitochondrial carrier family, is dependent on the guided entry of tail-anchored proteins (GET) complex. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. Loss of the GET pathway is detrimental to yeast cells experiencing mitochondrial import failure and prevents re-import of mitochondrial proteins from the ER via the ER-SURF pathway. Overall, this study outlines an important role for the GET complex in ER targeting of non-imported mitochondrial carrier proteins.

A Single Cysteine Residue in the Translocation Pathway of the Mitosomal ADP/ATP Carrier from Cryptosporidium parvum Confers a Broad Nucleotide Specificity

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes

Mitochondrial carrier family (MCF/SLC25) proteins are selective transporters that maintain the mitochondrial metabolome. Here we combine computational, biochemical and phenotypic approaches to understand substrate selectivity of SLC25A3. In mammals, SLC25A3 transports both copper and phosphate, yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2, which transports copper, and MIR1, which transports phosphate. To understand whether the ancestral state of this transporter was a single promiscuous transporter that duplicated and gained selectivity, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses reveal that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating that the gene duplication that created these paralogs occurred early in eukaryotic evolution. Frequent lineage-specific gene duplications and losses suggest that substrate specificity may be evolutionarily labile. To link this phylogenetic signal to protein function and resolve the residues involved in substrate selection, we used structural modelling and site-directed mutagenesis to identify PIC2 residues involved in copper and phosphate transport activities. Based on these analyses, we generated a Leu175Ala variant of mouse SLC25A3 that retains the ability to transport copper, but not phosphate, and rescues the cytochrome c oxidase defect in SLC25A3 knockout cells. Taken together, this work uses an evolutionary framework to uncover amino acids involved in substrate recognition by MCF proteins responsible for copper and phosphate transport.

The SLC25 Carrier Family: Important Transport Proteins in Mitochondrial Physiology and Pathology

Members of the mitochondrial carrier family (SLC25) transport a variety of compounds across the inner membrane of mitochondria. These transport steps provide building blocks for the cell and link the pathways of the mitochondrial matrix and cytosol. An increasing number of diseases and pathologies has been associated with their dysfunction. In this review, the molecular basis of these diseases is explained based on our current understanding of their transport mechanism.

Characterization of In Vivo Function(s) of Members of the Plant Mitochondrial Carrier Family

Although structurally related, mitochondrial carrier family (MCF) proteins catalyze the specific transport of a range of diverse substrates including nucleotides, amino acids, dicarboxylates, tricarboxylates, cofactors, vitamins, phosphate and H+. Despite their name, they do not, however, always localize to the mitochondria, with plasma membrane, peroxisomal, chloroplast and thylakoid and endoplasmic reticulum localizations also being reported. The existence of plastid-specific MCF proteins is suggestive that the evolution of these proteins occurred after the separation of the green lineage. That said, plant-specific MCF proteins are not all plastid-localized, with members also situated at the endoplasmic reticulum and plasma membrane. While by no means yet comprehensive, the in vivo function of a wide range of these transporters is carried out here, and we discuss the employment of genetic variants of the MCF as a means to provide insight into their in vivo function complementary to that obtained from studies following their reconstitution into liposomes.