Calcium regulation of mitochondrial carriers

    Calcium regulation of mitochondrial carriers Araceli del Arco, Laura Contreras, Beatriz Pardo, Jorgina Satrustegui PII: DOI: Reference:

S0167-4889(16)30077-5 doi: 10.1016/j.bbamcr.2016.03.024 BBAMCR 17839

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

25 January 2016 23 March 2016 23 March 2016

Please cite this article as: Araceli del Arco, Laura Contreras, Beatriz Pardo, Jorgina Satrustegui, Calcium regulation of mitochondrial carriers, BBA - Molecular Cell Research (2016), doi: 10.1016/j.bbamcr.2016.03.024

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ACCEPTED MANUSCRIPT Calcium regulation of mitochondrial carriers

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Araceli del Arco2,3,4, Laura Contreras1,3,4, Beatriz Pardo1,3,4, Jorgina Satrustegui1,3,4,* 1

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Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid‐Consejo Superior de Investigaciones Científicas, 28049, Madrid, Spain. 2 Facultad de Ciencias Ambientales y Bioquímica, Centro RegionaI de Investigaciones Biomédicas; Universidad de Castilla la Mancha, Toledo, 45071, Spain. 3 CIBER de Enfermedades Raras (CIBERER). 4 Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS‐FJD).

Highlights

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*Corresponding author: Jorgina Satrústegui Departamento de Biología Molecular Centro de Biología Molecular Severo Ochoa, c/ Nicolás Cabrera, 1. UAM. 28049-Madrid. Tel: 34-91-1964621 Fax: 34-91-1964420 Email: [email protected]; [email protected]

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Ca

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CaMCs comprise aspartate/glutamate (AGCs) and ATP-Mg/Pi (SCaMCs, APCs)

regulated mitochondrial carriers (CaMCs) transduce Ca2+ signals to mitochondria.

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2+

carriers 

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AGCs are active at basal Ca , and require small Ca

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These small Ca

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SCaMCs are activated by large Ca

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signals for further activation.

signals do not need to reach the mitochondrial matrix. 2+

signals that also reach the matrix.

Key words: Mitochondrial carriers; aspartate/glutamate carriers; ATP-Mg2+/Pi carriers; calcium; solute transport.

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ACCEPTED MANUSCRIPT Abstract Mitochondrial function is regulated by calcium. In addition to the long known effects of matrix Ca2+, regulation of metabolite transport by extramitochondrial Ca2+ represents an alternative

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Ca2+-dependent mechanism to regulate mitochondrial function. The Ca2+ regulated mitochondrial

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transporters (CaMCs) are well suited for that role, as they contain long N-terminal extensions

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harboring EF-hand Ca2+ binding domains facing the intermembrane space. They fall in two groups, the aspartate/glutamate exchangers, AGCs, major components of the NADH malate aspartate shuttle (MAS) and urea cycle, and the ATP-Mg2+/Pi exchangers or short CaMCs (APCs or SCaMCs). The AGCs are activated by relatively low Ca2+ levels only slightly higher than resting Ca2+, whereas all SCaMCs

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studied so far require strong Ca2+ signals, above micromolar, for activation. In addition, AGCs are not strictly Ca2+ dependent, being active even in Ca2+-free conditions. Thus, AGCs are well suited to

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respond to small Ca2+ signals and that do not reach mitochondria. In contrast, ATP-Mg2+/Pi carriers are inactive in Ca2+ free conditions and activation requires Ca2+ signals that will also activate the calcium uniporter (MCU). By changing the net content of adenine nucleotides of the matrix upon

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activation, SCaMCs regulate the activity of the permeability transition pore, and the Ca2+ retention

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capacity of mitochondria (CRC), two functions synergizing with those of the MCU. The different Ca2+

1. Introduction

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activation properties of the two CaMCs are discussed in relation to their newly obtained structures.

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In mitochondria the transport of metabolites, cofactors and nucleotides is performed by members of the mitochondrial carrier family (MCF or SLC25 family), the most expanded family of solute carriers in eukaryotes (reviewed in Palmieri, 2004; del Arco and Satrústegui, 2005; Palmieri, 2013). These proteins have a characteristic structure made up by three repeats of about 100 amino acids each formed by two transmembrane -helices connected by a long hydrophilic matrix loop with the N- and C-termini exposed to the cytosol (Saraste and Walker, 1982; Palmieri, 2004; Kunji 2004). MCF proteins present in the odd -helix a distinctive consensus motif which has facilitated largely their identification. This family contains members with a long N-terminal extension harbouring EF-hand Ca2+ binding domains of around 180 vs 330 residues, the L-CaMCs (for long Ca2+dependent Mitochondrial Carriers) which include Aralar also named Aralar1, the first to be identified as a protein with a mitochondrial localization (del Arco and Satrustegui, 1998), and the SCaMCs (for short Ca2+-dependent Mitochondrial Carriers), with a shorter N-terminal extension than the LCaMCs (Figure 1).

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ACCEPTED MANUSCRIPT The LCaMCs correspond to the mitochondrial transporters of aspartate/glutamate (AGC) (Palmieri et al., 2001). AGC catalyzes the electrogenic exchange of mitochondrial aspartate for cytosolic glutamate plus a H+ (LaNoue and Tischler, 1974; Palmieri et al., 2001), a critical step within

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the NADH malate-aspartate shuttle (MAS) and urea cycles. Although this transport activity was

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characterized biochemically much before the identification of the transporter proteins (LaNoue and Tischler, 1974), its regulation by calcium was unknown. In fact, reports of calcium regulation of MAS

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were attributed to matrix Ca2+ effects on -ketoglutarate dehydrogenase (Leverve et al., 1986; see Satrústegui et al., 2007a for review).

The SCaMCs (also named APCs) correspond to the mitochondrial transporters of ATP- Mg2+/Pi

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(Fiermonte et al., 2004; del Arco and Satrústegui, 2004). The existence of ATP-Mg2+/Pi transport activities in mitochondria modulated by cytosolic calcium signals was known prior to the identification of the transporters involved. In hepatocytes, glucagon treatment leads to an increase in

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adenine nucleotides (AdNs) content in mitochondria mediated by the rise in cytosolic Ca2+ produced by the hormone (Aprille et al., 1982; Haynes et al., 1986). A mitochondrial ATP-Mg2+/Pi transport activity, activated by increases in cytosolic Ca2+ and sensitive to calmodulin (CaM) antagonists, was

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identified as responsible for AdNs accumulation in liver mitochondria (Nosek et al., 1990; reviewed

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by Aprille, 1993). SCaMCs showed striking sequence homology at the N- and C-domain with those of CaM and ADP/ATP carrier (AAC), respectively, representing a suitable candidate for the liver ATP-

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Mg2+/Pi transporter (Fiermonte et al., 2004; del Arco and Satrústegui, 2004), as was indeed shown after reconstitution in proteoliposomes (Fiermonte et al., 2004). The classical pathway of calcium regulation of oxidative phosphorylation is thought to occur

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by calcium entry in mitochondria through the mitochondrial calcium uniporter (MCU) followed by the activation of the three matrix dehydrogenases of tricarboxylic acid cycle and complex V (McCormack et al., 1990; Glancy and Balaban, 2012). However, based on computer simulations, the existence of a parallel mechanism of activation by cytosolic factors has been suggested (Korzeniewski, 2007). Because EF-hand domains of CaMC N-extensions face the intermembrane space, these carriers were proposed to be regulated by cytosolic calcium (del Arco and Satrústegui, 1998; Palmieri et al., 2001), participating in the transduction of cytosolic Ca2+ signals to the mitochondrial matrix by the transport of a solute (reviewed in Satrústegui et al., 2007a). Consequently, CaMCs represent a mechanism to transduce calcium signals to mitochondria alternative to that of the MCU. CaMCs are widely distributed across all major branches of eukaryotic life. The genome of metazoa, plants, and most of fungi and protozoa usually have counterparts of both CaMCs (Cavero et al., 2003; del Arco and Satrustegui 2004; Monné et al., 2015; Lorenz et al., 2015, Harborne et al., 2015), indicating that transduction of Ca2+-signals through the transport activity of CaMCs could be an ancestral and evolutionary conserved mechanism. 3

ACCEPTED MANUSCRIPT From initial molecular identification of CaMCs, an intense work has been carried out in order to complete their characterization and to advance in the understanding of their physiological roles. Their role as mitochondrial metabolite carriers and as modulators of calcium signalling to

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mitochondria has been scrutinized (Palmieri et al., 2001; Lasorsa et al., 2003; Cavero et al., 2005;

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Satrústegui et al., 2007a; Traba et al., 2008; Mármol et al., 2009; Traba et al., 2012; Amigo et al., 2013; Llorente-Folch et al., 2013; Rueda et al., 2014; Rueda et al., 2015). However, the first insights

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concerning the mechanistic principles of their Ca2+-dependence have been only recently reached after obtaining of crystal structures of their regulatory Ca2+-binding domains (Thangaratnarajah et al., 2014; Yang et al., 2014; Harborne et al., 2015). In this review, we will attempt to summarize the

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knowledge acquired about both CaMC subfamilies highlighting their complexity along with their functional differences taking into account the structural data about their regulatory N-domains recently revealed. For further important aspects related to mitochondrial aspartate/glutamate and

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ATP-Mg/Pi carriers see Palmieri and Monné (2016).

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2. The mitochondrial aspartate/glutamate carriers There are two isoforms of the mitochondrial AGC in humans, Aralar/AGC1/SLC25A12 and

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citrin/AGC2/SLC25A13 (del Arco and Satrustegui, 1998; del Arco et al., 2000; Palmieri et al., 2001; del Arco et al., 2002; Contreras et al., 2007). Both isoforms are 77 % identical showing highest homology

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with the mitochondrial glutamate carriers (GC1 and GC2; Fiermonte et al., 2002). The AGC catalyzes the electrogenic exchange of mitochondrial aspartate for cytosolic glutamate plus a H+, a critical step within the NADH malate-aspartate shuttle (MAS) and urea cycles (LaNoue and Tischler, 1974;

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Palmieri et al., 2001). Due to its membrane potential dependence, this transport is irreversible under physiological conditions and hence a potential site for regulation. In isolated mitochondria, the AGC is also required for respiration on glutamate plus malate (Jalil et al., 2005). Accordingly, overexpression of AGC isoforms in cell lines increase MAS activity (Palmieri et al., 2001; Rubi et al., 2004). AGC/MAS are critical in cells that oxidize glucose in mitochondria. Studies in intact neurons from AGC1-KO mice using glucose as only substrate have shown that basal respiration is reduced by around 40 %, indicating that the shuttle is essential for the oxidation of glucose (Llorente-Folch et al., 2013). S. cerevisiae has one single mitochondrial aspartate/glutamate carrier, agc1p. This transporter functions within a malate aspartate shuttle which is required for growth on acetate and fatty acids as carbon sources (Cavero et al., 2003).). In human and rodents, AGC isoforms show a divergent distribution pattern. Aralar/AGC1 is preferentially expressed in excitable tissues as brain and skeletal muscle whereas high expression level of AGC2/citrin is detected in liver, kidney and heart (Kobayashi et al., 1999; del Arco et al., 2000; 4

ACCEPTED MANUSCRIPT del Arco et al., 2002; Ramos et al., 2003). These divergent patterns match the phenotypes observed in AGC-deficient mice and those associated to human diseases caused by AGC mutations (Kobayashi et al., 1999; Saheki and Kobayashi, 2002; Jalil et al, 2005; Wibom et al., 2009; Falk et al., 2014).

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Mutations in AGC2/citrin cause disorders of the urea cycle, type II citrullinemia (CTLN-2) and

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a neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) (Kobayashi et al., 1999; Saheki and Kobayashi, 2002). In these urea cycle diseases failure to export aspartate out of the liver

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mitochondria results in an instability of the aspartate and citrulline utilizing enzyme argininosuccinate synthetase, leading to elevated citrulline levels. The relevance of citrin/AG2-MAS as liver NADH shuttle is much higher in human than in mice and only a double KO mouse lacking both

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citrin/AGC2 and mitochondrial glycerol-phosphate dehydrogenase, the other major NADH shuttle in liver, but not the single citrin KO mouse, fully displays features of human citrin deficiency (Saheki et al., 2007; Saheki et al., 2011).

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On the other hand, mutations in Aralar/AGC1 cause congenital hypotonia, psychomotor developmental arrest and seizures, global cerebral hypomyelination (OMIM:612949) and reduced Nacetylaspartate (NAA) consistent with defects in energy metabolism in brain, and elevated plasma

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lactate levels (Wibom et al., 2009; Falk et al., 2014). The Aralar/AGC1 KO mice also recapitulates

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many phenotypic features of human AGC1-deficiency (Jalil et al., 2005; Sakurai et al., 2010; Ramos et al., 2011; Gómez-Galán et al., 2012; Pardo et al., 2011). In addition, studies in Aralar-KO mice have

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led to the discovery of new roles for this carrier demonstrating that Aralar/AGC1 is required for synthesis of brain aspartate and N-acetylaspartate, a neuron-born metabolite that supplies acetate to oligodendrocytes for yet unknown functions one of which may be myelin lipid synthesis

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(Satrústegui et al., 2007a; Satrústegui et al., 2007b; Ramos et al., 2011; Wibom et al., 2009; Maier et al., 2015). The critical role of AGC1 in producing neuronal aspartate for the brain once the blood brain barrier has been established is probably the major cause of the altered brain development in AGC1 deficiency. This role has revealed new aspects of inter- and intracellular traffic of amino acids and glutamine synthesis in brain. Thus, the efflux of aspartate from neurons to astrocytes is required for astrocytic de novo glutamate synthesis and subsequent glutamine formation (Pardo et al., 2011). This same transcellular pathway from photoreceptors to Müller glial cells operates in retina (Du et al., 2013; Lindsay et al., 2014). On the other hand, Aralar/AGC1/SLC25A12 has also been identified as a susceptibility gene for autism spectrum disorders (ASD) by association studies (reviewed by Napolioni et al., 2011), apparently through an upregulation of AGC1 levels. 3. The mitochondrial ATP-Mg2+/Pi carriers

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ACCEPTED MANUSCRIPT In mammals, there are five isoforms of the ATP-Mg2+/Pi carrier; SCaMC-1/SLC25A24, SCaMC2/SLC25A25, SCaMC-3/SLC25A23, SCaMC-1L/Slc25a54 and SCaMC-3L/SLC25A41 (also named APCs for “ATP-Mg2+ Phosphate Carrier”, Fiermonte et al., 2004). All code for highly conserved proteins (about 65-80% identity) and, except SCaMC-3L/SLC25A41, all are about 500 amino acids with a N-

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terminal harboring EF-hand Ca2+-binding motifs homologous to CaM (Fiermonte et al., 2004; del Arco and Satrustegui, 2004). The fifth isoform SCaM-3L/slc25a41 lacks of N-extension (Traba et al., 2009).

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This structural complexity has arisen through gene duplications during the evolution of vertebrates (Traba et al., 2009; Amigo et al., 2012) and in mammals it is increased by the existence of tissuespecific splice variants (del Arco and Satrustegui, 2004; del Arco, 2005; Satrustegui et al., 2007a).

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SCaMC-1/Slc25a24 corresponds to EFINAL, a protein present in intestine and initially described as peroxisomal (Weber et al., 1997).

ATP-Mg2+/Pi carriers perform the electroneutral exchange of ATP-Mg2+ (MgATP2−) or ADP

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(HADP2-) for Pi (HPO42−) across the mitochondrial inner membrane (Fiermonte et al., 2004,), thereby controlling the net levels of AdNs (AMP+ADP+ATP) in the matrix (Joyal and Aprille, 1992), representing an alternative pathway for mitochondrial nucleotide transport to the AAC which

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performs a strict ADP/ATP counter-exchange (Traba et al., 2008). In addition, it has been reported

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that ATP-Mg2+/Pi carriers from Arabidopsis thaliana reconstituted into liposomes can also transport AMP, adenosine 5'-phosphosulfate or ATP-Ca2+ (Monné et al., 2015; Lorenz et al., 2015) and can also

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perform a counter-exchange of AdNs against sulfate or thiosulfate (Monné et al., 2015). The amino acids responsible for selectivity for ATP-Mg2+ rather than ATP at the substrate binging site have been recently identified (Run et al., 2015).

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By modulating the AdNs content in mitochondria, it was proposed that the ATP-Mg2+/Pi carrier could affect relevant cellular functions as respiratory activity or mitochondrial biogenesis as well as the activity of matrix ATP-dependent process as gluconeogenesis or urea synthesis in liver (reviewed in Aprille, 1993). In the last years, different loss of function models have been developed providing relevant data about its function. In S. cerevisiae the simultaneous absence of ATP-Mg2+/Pi carrier and AAC2, the major isoform of AAC carrier in yeast, is lethal revealing a functional interplay between both AdNs carriers (Chen et al., 2004; Traba et al., 2008; Traba et al., 2009). In mammals, SCaMC-1/SLC25A24 is abundantly expressed in tumor cells and embryonic tissues, SCaMC-3/SLC25A23 is preferentially, but not exclusively, expressed in liver and brain and SCaMC-2/SLC25A25 in skeletal muscle and brain (del Arco and Satrústegui, 2004; Satrústegui et al., 2007a; Traba et al., 2012). SCaMC-3L/slc25a41 and SCaMC-1L/slc25a54 expression apears restricted to testis in mice (Traba et al., 2009; Amigo et al., 2012). Their redundancy along with their overlapping expression patterns could be the origin of the absence of disease-causing mutations associated to human ATP-Mg2+/Pi carrier genes. Besides, the deficient-mouse models developed for 6

ACCEPTED MANUSCRIPT SCaMC-1/SLC25A24, SCaMC-3/SLC25A23 and SCaMC-2/SLC25A25 are all viable showing only phenotypic manifestations in those tissues where they are the major isoforms (Anunciado-Koza et al., 2011, Amigo et al., 2013; Rueda et al., 2015, Urano et al., 2015). However, regardless of the

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absence of gross phenotypic abnormalities their study has allowed to decipher specific functional

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roles for each isoform (Anunciado-Koza et al., 2011, Amigo et al., 2013; Rueda et al., 2015). In liver mitochondria, SCaMC-3/SLC25A23-dependent accumulation of AdNs in AdN-depleted liver

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mitochondria is required to acquire a fully active state 3 respiration, although further accumulation of AdNs is not followed by increases in respiration (Amigo et al., 2013). SCaMC-3/SLC25A23 is also necessary for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca2+-mobilizing agents, probably favoring a Ca2+-dependent accumulation of

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mitochondrial AdNs and matrix Ca2+ (Amigo et al., 2013). Similarly, in the absence of SCaMC2/SLC25A25 the efficiency of ATP production required for skeletal muscle function is also diminished

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(Anunciado-Koza et al., 2011). SCaMC-2/SLC25A25-deficient mice under high energetic requirements, such as those produced during forced treadmill exercise, showed a reduced physical endurance

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4. Ca2+-activation of CaMCs

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(Anunciado-Koza et al., 2011).

CaMC subfamilies display EF-hands domains able to bind calcium, as determined by 45Ca2+

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overlay assays (Table 1), and their N-domains harbor sequences unrelated to each other which are thought to account for the distinctive responses to cytosolic Ca2+ signals of different family members (summarized in Table 1) (Figure 1). In AGCs, the sequences of their N-terminal extension and the

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arrangement of the EF-hands have no homologs within the family of Ca2+-binding proteins. Using hidden Markov models, nine different EF-hands were predicted at the first 330 residues of Aralar/AGC1 and citrin/AGC2 (Contreras et al., 2007), although the ninth EF-hand (positions 302–330 in human Aralar/AGC1), is not equally consistent (Contreras et al, 2007, Thangaratnarajah et al., 2014). It was suggested that EF-1 and EF-2 were active in both Aralar/AGC1and citrin/AGC2, but that EF-4, EF-6, EF-7 and EF-9 were nonfunctional for Ca2+-binding (Contreras et al., 2007). This unequal contribution of the EF-hands to Ca2+-binding has been confirmed recently by structural studies (see below) (Thangaratnarajah et al., 2014). EF-hand assortment is conserved between AGC orthologs with the exception of the yeast AGC ortholog, Agc1p, which does not have Ca2+-binding motifs in its long N-extension indicating that its activity is not Ca2+-dependent (Cavero et al., 2003). Notably, S. cerevisiae is an exception in terms of Ca2+ signaling in mitochondria since it does not have an MCU ortholog or Ca2+-sensitive TCA dehydrogenases (Satrústegui et al., 2007a). Ca2+ activation of MAS activity in isolated brain and heart mitochondria showed that AGCs were stimulated by Ca2+ concentrations below micromolar (Table 1) (Pardo et al., 2006; Contreras et 7

ACCEPTED MANUSCRIPT al., 2007). MAS activity in brain mitochondria, where Aralar/AGC1 is the main isoform (Contreras et al., 2010), was stimulated around 3-fold by extramitochondrial Ca2+ with a half-maximal activation (S0.5) of ~300 nM (Pardo et al., 2006; Contreras et al., 2007) and Ca2+-activation of respiration of brain

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mitochondria on glutamate plus malate showed a similar Ca2+ requirement (Gellerich et al., 2009). However, in liver mitochondria, with citrin/AGC2 as main isoform, MAS activity was increased about

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1.5-fold in response to Ca2+ with an S0.5 for Ca2+ activation of ~150 nM (Contreras et al., 2007),

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suggesting a fully activated transporter activity under liver resting conditions. It should be mentioned that the other main NADH shuttle, the glycerophosphate shuttle is also activated by calcium on the external side of the inner mitochondrial membrane, through Ca2+-binding EF-hands facing the intermembrane space (reviewed in Satrustegui et al., 2007a).

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The affinity for Ca2+ of AGC1 is higher than that of the Ca2+ uniporter (Ca2+ concentrations above micromolar), suggesting that MAS may be activated by Ca2+ signals that do not reach the

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mitochondrial matrix. Activation by Ca2+ of mitochondrial function has been shown to occur in neurons, independently of the effects of Ca2+ on workload, i. e., strictly via the regulatory role of calcium on mitochondrial dehydrogenases and/or complex V, or through extramitochondrial effects

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on the CaMCs (Llorente-Folch et al., 2013). Interestingly, small calcium signals such as those provided

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by the activation of G protein coupled receptors result in a fully AGC1-dependent and Ca2+dependent activation of respiration and NADH transfer to mitochondria in intact neurons (Pardo et

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al., 2006; Llorente-Folch et al., 2013), excluding a relevant role of the MCU under these conditions. In fact, the increase in neuronal respiration in response to moderate Ca2+ signals and workloads (as those brought about by K+-depolarization), is also strictly dependent on AGC1, which also plays an

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important role in the response to large workloads (veratridine). By shifting pyruvate away from lactate and into mitochondria, MAS increases pyruvate utilization in a Ca2+-dependent way, so that the impaired respiratory responses in AGC1-deficient neurons are all rescued by the provision of exogenous pyruvate (Llorente-Folch et al., 2013, Gellerich et al., 2012; 2013; Rueda et al., 2014; Llorente-Folch et al., 2015). On the other hand, large calcium signals that enter the matrix and activate mitochondrial dehydrogenases, particularly α-ketoglutarate dehydrogenase, may result in inhibition of MAS, not via effects on AGC1, but through inhibition of the 2-oxoglutarate carrier (OGC), the other transporter component of MAS (Contreras and Satrústegui, 2009). In neurons, but not in liver, this may lead to an inhibition of MAS which would reverse upon deactivation of the matrix dehydrogenase (Pardo et al., 2006, Contreras and Satrústegui, 2009; Satrústegui and Bak, 2015). SCaMCs display a shorter N-domain, 160-180 residues (Table 1), with a relevant homology to CaM,  50 % of similarity in the amino acid sequences (del Arco and Satrústegui, 2004), and totally unrelated to that of AGCs. SCaMC N-domain exhibit a distribution of EF-1/EF-2 and EF-3/EF-4 pairs 8

ACCEPTED MANUSCRIPT similar to that of CaM, except for the flexible linker at the central region that connects both EF pairs, which is shortened in SCaMC isoforms (del Arco and Satrústegui, 2004; Harborne et al., 2015). This smaller linker domain is distinctive of SCaMCs subfamily since it is not present in other CaM-like proteins.

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In yeast and mammalian SCaMC orthologs, Ca2+-dependence of transport were determined in experiments of AdNs transport using isolated mitochondria or permeabilized cells expressing

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mitochondrial targeted Luciferase (Cavero et al., 2005; Traba et al., 2008; Traba et al., 2009; Traba et al., 2012; Amigo et al., 2013). In contrast to AGCs, SCaMCs showed S0.5 values for Ca2+ activation of transport in the μM range, from 3 to 15 μM (Cavero et al., 2005; Traba et al., 2008; Traba et al., 2012; Amigo et al., 2013) (see Table 1), substantially higher than the values showed by AGCs. These values

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also contrast with those described for the A. thaliana orthologs with S0.5 for Ca2+ activation ranging from 0.2 to 0.8 μM as determined in transport assays of recombinant proteins in proteoliposomes

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(Monné et al., 2015). These lower values have been associated with the lower Ca2+ concentration present in the cytoplasm of plant cells (Monné et al., 2015). Interestingly, these transport assays have also revealed that the activity of ATP-Mg2+g/Pi carriers was strictly Ca2+-dependent, remaining

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totally inactive below M Ca2+ (Haynes et al., 1986; Traba et al., 2008; Traba et al., 2009; Amigo et al.,

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2013). Their complete dependency for Ca2+ contrasts with that of AGCs, which show transport activity in the absence of Ca2+ (Contreras et al., 2007). Of note, the Ca2+ sensitivity of the SCaMCs is

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close to that of the MCU (apparent Km about 10-20 μM under physiological conditions (Kirichok et al., 2004)), and also close the Ca2+ concentrations those present in ER-Mitochondria microdomains, MAMs (Giacomello et al., 2010). This suggests that these carriers will be activated when the MCU is

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also activated and may synergize with the MCU in capturing Ca2+. Indeed, the results obtained in SCaMC-3 KO neurons undergoing glutamate excitotoxicity (Rueda et al., 2015) strongly support this possibility. This is also the case for SCaMC-1-silenced cells undergoing a Ca2+ challenge (Traba et al., 2012). SCaMCs induce an uptake or efflux of AdNs in mitochondria when activated by strong Ca2+ signals, which results in modulation of mitochondrial AdNs. By doing so, SCaMC activity increases the Ca2+ retention capacity (CRC) of mitochondria (Traba et al., 2012; Amigo et al., 2013; Rueda et al., 2014; Rueda et al., 2015). It is interesting to note that ATP or ADP concentrations required for uptake along the SCaMCs are fully physiological, so that the whole process will take place in any cell provided that a strong Ca2+ signal activates SCaMC. Although the exact explanation of the increase in CRC caused by the ATP-Mg2+/Pi carrier is yet to be clarified, one possibility is that the presence of AdNs in the matrix stabilizes Pi-Ca2+ complexes known to buffer matrix Ca2+, resulting in lower matrix free Ca2+ levels (Nicholls and Chalmers, 2004; Kristián et al., 2007). Indeed, SCaMC-1 deficiency results in higher matrix Ca2+ signals in response to a cytosolic Ca2+ transient (Traba et al., 2012) and 9

ACCEPTED MANUSCRIPT SCaMC-3 deficiency also results in higher matrix Ca2+ in response to NMDA-induced Ca2+ signals (Rueda et al., 2015). Another possibility relates to the well-known inhibitory effects of AdNs on the permeability transition pore (PTP) (reviewed by Bernardi, 1999) that opens in response to the same

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Ca2+ signals that activate the MCU and the SCaMCs. In other words, by increasing matrix AdNs under conditions in which the MCU is operating, SCaMC1 in cancer cells and SCaMC-3 in hepatocytes and

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neurons lower matrix Ca2+ and desensitize PTP opening to Ca2+ (Traba et al., 2012). Silencing of

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SCaMC-1/SLC25A24 in tumor cells resulted in a drastic reduction of mitochondrial CRC, and sensitization to mitochondrial PTP mediated necrotic death triggered by oxidative stress caused by H2O2 or menadione treatment which cause Ca2+ overload (Traba et al., 2012). Knockout of SCaMC-

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3/slc25a23 in brain also increases the susceptibility the toxic effects of glutamate (Rueda et al., 2015). SCaMC-3/slc25a23-deficiency decreases the CRC of brain mitochondria exposed to mM [AdNs], hastens the appearance of delayed Ca2+ deregulation and neuronal death in response to

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excitotoxic glutamate or NMDA concentrations, and causes a larger seizure response to Kainate (Rueda et al., 2015). Interestingly, these effects of SCaMC-3/slc25a23 deficiency on neuronal survival are secondary to an earlier and rapid effect on respiration. In deficient neurons, excitotoxic NMDA

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concentrations induce a rapid increase in PARP-1 activity which causes a drop in matrix ATP levels

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and thereby prevents a complete up-regulation of respiration. Therefore SCaMC-3/slc25a23, by taking up AdNs, overrides the drop in matrix ATP and allows a full up-regulation of respiration upon

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NMDA addition (Rueda et al., 2015; reviewed in Rueda et al 2016 submitted to BBA). Hoffman and coworkers (Hoffman et al., 2014), have also observed a stimulatory effect of SCaMC-3/SLC25A23 on Ca2+ uptake in mitochondria through MCU, using HeLa cells silenced for the

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ATP-Mg2+/Pi carrier, which match the fall in CRC of mitochondria in tissues from SCaMC-3 KO mice (Amigo et al., 2013; Rueda et al., 2015). Hoffmann et al. also reported an interaction of SCaMC3/SLC25A23 with MCU and MICU1, a regulatory component of the MCU complex. However, other findings in SLC25A23-silenced cells contrast with those of SCaMC-3 KO tissues. For example, in contrast with the increased decline in cytosolic ATP levels and increased susceptibility to cell death observed in SCaMC-3 KO neurons, SCaMC-3/slc25a23 knockdown HeLa cells showed an attenuated oxidant-induced ATP decline and reduced cell death (Hoffman et al., 2013). The reasons for this discrepancy are yet unknown. 5. Structural basis for the mechanism of Ca2+ activation of CaMCs In the last years, the resolution of the tridimensional (3D) structure of the bovine AAC in complex with its cytosolic inhibitor carboxyatractyloside has allowed to propose a common mechanism for solute translocation by MCF Proteins (Pebay-Peyroula et al., 2003; Robinson and Kunji, 2006; Robinson et al., 2008). During transport cycle, the binding of substrate to specific 10

ACCEPTED MANUSCRIPT residues placed in its central cavity causes conformational changes that favor its opening (Nury et al., 2006; Robinson and Kunji, 2006; Robinson et al., 2008). In CaMCs, therefore, Ca2+-binding to the Ndomain must control the accessibility of solutes to the substrate binding site from cytosol and its

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efflux from matrix side. Recently, crystal 3D structures for the N-domains of both CaMCs have also been solved providing important clues to explain how the access to the translocation pathway is

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differentially modulated by Ca2+ in these carriers (Thangaratnarajah et al., 2014; Yang et al., 2014;

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Harborne et al., 2015).

5.1 Structural basis for the mechanism of Ca2+ activation of SCaMCs

Two equivalent 3D structures have been solved of the human SCaMC-1/SLC25A24 N-domain, amino

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acids 1-194 (Yang et al., 2014) and 14-174 (Harborne et al., 2015), in a Ca2+-bound state. The structures solved have indicated that the regulatory N-domain functions as a monomer. In both

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structures, the N-domain showed a compacted architecture in which, as suggested from its similarity with CaM, all four EF-hands have the typical conformation described for Ca2+-bound CaM (Yang et al., 2014; Gifford et al., 2007) but organized in a different assortment. Thus, the absence of the flexible

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linker distinctive of CaM-like proteins that connects the EF-1/EF-2 and EF-3/EF-4 pairs produces a

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rigid structure where EF pairs are positioned in proximity establishing extensive hydrogen bonds and nonpolar interactions (Yang et al., 2014; Harborne et al., 2015) (Figure 2A). In addition, the N-domain

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presents a C-terminal amphipathic α-helix, residues 159-168, conserved among SCaMC isoforms, that forms nonpolar interactions with the hydrophobic cleft of the EF-3/EF-4 pair participating in Ca2+regulation (-H9, Figure 2A). Interestingly, this C-terminal α-helix in complex with the N-domain resembles the target peptide in several CaM-peptide complexes (Hoeflich and Ikura, 2002), and it has

2014).

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been proposed that it serves as an internal target peptide for the N-domain EF-hands (Yang et al.,

Despite their high sequence similarity, SCaMCs show N-terminal variants, generated by alternative exons 1, which lack EF-1, SCaMC-2a/SLC25A25, or contain non-functional EF-1 hands, SCaMC-2b/SLC25A25 and SCaMC-2c/SLC25A25, and could show an altered regulation by calcium (Figure 2B) (del Arco and Satrústegui, 2004; Fiermonte et al., 2004; Satrústegui et al., 2007a). Furthermore, in SCaMC-2/SLC25A25 its complexity appears increased by the existence of additional splice variants generated by the inclusion of an evolutionarily conserved micro-exon (Figure 2B). Recently, two independent reports have identified these variants as belonging to a set of microexons regulated by neural-specific splicing factors (Irimia et al., 2014; Li et al., 2015). Notably, these micro-exons encode domains important for protein-protein interactions and it has been predicted that their inclusion in neurons may modulate protein interactions relevant for neural functions (Irimia et al., 2014; Li et al., 2015). In SCaMC-2/SLC25A25 isoforms, the micro-exon (micro-exon 3b in 11

ACCEPTED MANUSCRIPT Figure 2B) introduces 12 residues between the entering -helix 7 and the EF-loop, generating a longer -helix that disrupts the EF-4 hand (Figure 2B). As a consequence, the conformation adopted by the C-terminal EF-3/EF-4 pair can be changed altering its functionality and, therefore, the

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inclusion of the micro-exon could increase the functional repertoire of the regulatory N-domain.

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Unfortunately, no data are available on Ca2+ activation of SCaMC-2/SLC25A25 variants. Because the 3D structures of SCaMC-1/SLC25A24 N-domain in Ca2+-free state have not been

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achieved, alternative approaches have been used to explore the potential Ca2+-triggered conformational changes in this regulatory domain. Models of the SCaMC-1/SLC25A24 N-domain in the Ca2+-free state have been generated based on structures of related EF-hand proteins (Harborne

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et al., 2015). In addition, the structural properties of the N-domain in the Ca2+-free and Ca2+-bound state have been analyzed by NMR spectroscopy revealing the existence of substantial structural

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differences between them (Yang at el., 2014). Notably, both approaches showed coincident results indicating that upon release of calcium the C-terminal amphipathic α-helix does not interact with the EF-3/EF-4 pairs. Two alternative mechanisms have been suggested to explain in what manner this

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conformational change regulates the activity of the carrier (Figure 3A). Harborne and co-workers

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propose a mechanism where the amphipathic -H9 becomes mobile upon release of calcium and could block the transport of metabolites (Figure 3A (ii)) (Harborne et al., 2015) whereas that Yang

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and co-workers have suggested that in this situation the N-domain adopts a dynamic and loose conformation that interacts with the C-terminal domain causing it to close (Figure 3A (i)) (Yang et al., 2014).

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Notably, both models are coincident in that when calcium is not bound to the N-domain the carrier remains fully inactive, as has repeatedly been reported for the ATP-Mg2+/Pi carrier activity (Traba et al., 2008; Traba et al., 2009; Amigo et al., 2013). These structural findings also corroborate previous data obtained with mutated EF variants of SCaMCs. Thus, in Sal1p, the yeast ortholog of SCaMC, mutations in a single EF-domain, the canonical EF-2 or EF-3 hand, that abolish its ability to bind 45Ca2+ generate non-functional variants as indicated by failure to rescue the lethal phenotype of aac2/sal1Δ double mutants described for wild type Sal1p (Chen, 2004). Similarly, the introduction of mutations designed to abolish Ca2+ binding to EF-1 or EF-3 in the human SLC25A23/SCaMC-3 isoform affects its functionality (Hoffman et al., 2014). These results also suggest that, as described for CaM (Gifford et al., 2007), the EF-pairs of the N-domain could bind Ca2+ with positive cooperativity. 5.2 Structural basis for the mechanism of Ca2+ activation of AGCs

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ACCEPTED MANUSCRIPT The N-terminal domain of Aralar/AGC1, both in Ca2+-bound and Ca2+-free state, and the Nterminal domain fused to the C-terminal 60 amino acids of citrin/AGC2, only in Ca2+-bound state, have been crystallized and their structures solved to resolution 2.3, 2.4 and 2.4 Å, respectively,

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providing relevant and, a priori unexpected, findings (Thangaratnarajah et al., 2014). Thus, in the

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solved structures, the AGC N-domains can form homodimers where the EF-hand motifs are arranged in a new manner forming an arch in which EF-4–8 create a dimerization interface whereas EF-1–3 are

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located on the periphery participating in the regulation by Ca2+, although only EF-2 would bind Ca2+ in Aralar/AGC1 and citrin/AGC2 N-terminal domains. Interestingly, in the Ca2+-free state the entering and exiting -helices of EF-2 adopt a conformation similar to that of Ca2+-bound state. In preformed

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EF-hands the entropic cost of EF-loop ordering is decreased (Gifford et al., 2007) and it contributes to the high Ca2+ affinity found in both AGC isoforms (Pardo et al., 2006; Contreras et al., 2007; Gellerich et al., 2009). Interestingly, purified full-length citrin/AGC2 and shortened N-domain versions of both

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AGCs showed by chromatography analysis a size twice the theoretical mass supporting that AGCs can form dimers mediated by their N-domains (Thangaratnarajah et al., 2014). The possibility of dimerization by AGCs was totally unanticipated because in the last years it was well established from

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structural and functional analysis that MCF proteins function as monomers (revised by Kunji and

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Crichton, 2010). However, only the N-domains are involved in dimerization, the transport domains remain as independent units (Figure 3B). If dimerization is really functional in vivo is actually not

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known, although some evidences suggest that it could, in fact, be required for transport activity. Thus, the overexpression of truncated AGCs variants lacking the N-domain failed to increase mitochondrial ATP levels in response to Ca2+-mobilizing agents as was observed with full versions of

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AGCs (Lasorsa et al., 2003). In addition, a mutation in EF-4, in a residue proposed as participant in the dimerization surface, G176V, has been found associated with citrin/AGC2 deficiency (Song et al., 2013).

The superposition of Ca2+-bound and Ca2+-free states of the N-domains has allowed to Thangaratnarajah and coworkers to propose a model for AGCs activation where Ca2+-binding to EF-2 causes the opening of a cavity proximal to the carrier domain that acts as a vestibule to allow the access of the substrates (Thangaratnarajah et al., 2014). The conformational change from the Ca2+bound state to Ca2+-free state involves movements of the mobile unit of EF-hands 1–2 respect to the static region formed by EF-hands 4–8, a rotation of 42 degrees with the axis between EF-2/EF-3 (Thangaratnarajah et al., 2014). This movement favors interactions between residues located in the loops that connect EF-3 with EF-4 and EF-4 with EF-5 and as result closes a cavity in the regulatory Ndomain that may limit the access of the substrates to the carrier domain. Therefore, in contrast to SCaMCs, AGCs in its Ca2+-free state are still active for solute transport (figure 3C).

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ACCEPTED MANUSCRIPT Although data from mutational approaches on EF-hand motifs of AGCs are limited, some evidences have confirmed the distinctive role of EF-1 and EF-2 in the activation by Ca2+ of the Ndomain. In INS-1 -cells the expression of a double EF-1/EF-2 mutant form of human Aralar/AGC1

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eliminated Ca2+ activation of MAS by cytosolic Ca2+ signals, while that its basal activity was

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maintained (Mármol et al., 2009). Similarly, by complementation assays in AGC-deficient yeast, both WT and EF-1/EF-2 mutant versions of human citrin/AGC2 can revert its defect of growth on acetate

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as a carbon source, although the EF-1/EF-2 mutant form showed a reduced growth, around 50 %, respect to control (Wongkittichote et al., 2013). Interestingly, citrin/AGC2 harbouring mutations in either a single EF-hand or both result in similar growth rates, indicating that the inactivation of a

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single EF-hand of the EF-1/EF-2 pair is sufficient to eliminate Ca2+-activation in AGCs. Accordingly, deletion of EF-1 in citrin/AGC2 causes loss of Ca2+ binding ability (del Arco et al., 2000). In citrullinemia patients, mutations in the entering -helix of EF-1, A25E, and the exiting -

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helix of EF-2, L85P, have been associated to NICCD (Fu et al., 2011; Dimmock et al., 2009) and their pathogenic character has been attributed to a lack of competence for transport rather than with Ca2+-regulation as such. Thus, mutation L85P has been described in a compound heterozygote which

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does not show citrin/AGC2 protein in liver, indicating that this mutation leads certainly to a

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citrin/AGC2 deficiency (Fu et al., 2011). In contrast, mutation A25E has been detected in a homozygous patient which showed no alterations in citrin/AGC2 levels (Dimmock et al., 2009). This

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substitution might affect carrier functionality because Ala25 participates in stabilising interactions

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with the C-terminal residues (Thangaratnarajah et al., 2014).

Legends to figures

Figure 1. Comparison of the CaMCs structures. A schematic diagram of the domain organization of AGCs and SCaMCs is depicted. Proteins are drawn to scale. EF, EF-hand calcium binding domains; MC repeat, tandem repeats characteristic of mitochondrial carriers; CTD, Carboxy-terminal domain presents only in AGCs. EF-hand domains able to bind Ca2+ are indicated, the calcium ions are marked as green spheres. Figure 2. A) Cartoon representation of Ca2+-bound N-domain of SCaMC-1/SLC25A24 (PDB ID: 4N5X; Yang et al., 2014) with residues of Ca2+ binding sites in stick representation and Ca2+ ions as spheres. The figure was generated using PyMOL. The regulatory extra -helix (-H9) of N-domain is indicated. B) Schematic representation of SCaMC-2/SLC25A25 N-domain variants. The genomic organization of the 3’ region containing the alternatively spliced exons is shown at the top. For each SCaMC-

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ACCEPTED MANUSCRIPT 2/SLC25A25 variant, the EF hands (EF-1–4) with its eight α-helices (α1–8) and the additional -helix 9 (-9) are shown. Non-functional EF-1 hands encoded by alternative exons 1 (yellow) are marked by asterisk and disrupted EF-4 hands as EF-4. The residues derived from micro-exon 3b* are colored in

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green. The length of the N-domain of each SCaMC-2/SLC25A25 variant is also indicated.

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Figure 3. Proposed models for the mechanisms of calcium regulation of CaMCs

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A,B) Proposed mechanisms for SCaMCs, the mitochondrial ATP-Mg2+/Pi carriers. (A) In the presence of M [Ca2+], four Ca2+ ions bind to the EF-hands at the CaM-like regulatory N-domain which adopts in its calcium-bound state a rigid structure that allows the access of the solutes to substrate binding site inside the translocation domain (A) The N-domain is represented according to the SCaMC-

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1/SLC25A24 Ca2+-bound N-domain 3D structure used in figure 2. The substrate-binding site is shown in the carrier domain. (B) When cytosolic [Ca2+] is below the M range the regulatory N-domain does

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not bind Ca2+ and the carrier remains completely inactive. Two models have been proposed to explain how the regulatory N-domain closes the carrier in the Ca2+-free state; (I) in the apo state the N-domain has a disordered structure and acts as a cap that physically blocks substrate transport

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(Yang et al., 2014) and (II) in Ca2+-free state the -H9 is displaced hindering the pass of the solutes

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through the translocation pathway (Harborne et al., 2015). C,D) Proposed mechanism of calcium regulation of the mitochondrial aspartate/glutamate carriers.

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Diagrams of the Aralar/AGC1 in the Ca2+-bound state (C) and the apo state (D) at the same view based on the structures solved for them (PDB ID: 4P5X and PDB ID: 4P60, respectively; Thangaratnarajah et al., 2014). In both structures the N-domain of Aralar/AGC1 has been found as a

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dimer maintained by interactions between the EF-4/EF-8 regions of each monomer. EF-1-3 hands are highlighted in the picture. Ca2+-binding to EF-2, when cytosolic [Ca2+] increases from 0.3 M, or its dissociation in conditions of cytosolic resting [Ca2+] cause conformational changes at the mobile sub-unit formed by EF-hands 1–2 that originate a further opening or closing of a hydrophobic groove in the N-domain favouring, Ca2+bound-state (C), or limiting, apo-state (D), the access of the substrates to the carrier domain.

Acknowledgements This work was supported by Ministerio de Economía y Competitividad Grant BFU2011-30456 (to J. S.) and SAF2014-56929R (to J. S. and B. P.), by Centro de Investigación Biomédica en Red de Enfermedades Raras [an initiative of the Instituto de Salud Carlos III (ISCIII)], by Comunidad de

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ACCEPTED MANUSCRIPT Madrid Grant S2010/BMD-2402 MITOLAB-CM (to J.S.) and by an institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa.

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References

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Amigo I, Traba J, Satrústegui J, del Arco A (2012) SCaMC-1Like a Member of the Mitochondrial Carrier (MC) Family Preferentially Expressed in Testis and Localized in Mitochondria and Chromatoid Body. PLoS One 7:e40470.

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Amigo I, Traba J, Gonzalez-Barroso MM, Rueda CB, Fernandez M, Rial E, Sanchez A, Satrustegui J, del Arco A (2013) Glucagon Regulation of Oxidative Phosphorylation Requires an Increase in Matrix Adenine Nucleotide Content through Ca2+ Activation of the Mitochondrial ATP-Mg/Pi Carrier SCaMC-3. J. Biol. Chem. 288:77917802.

MA

Anunciado-Koza RP, Zhang J, Ukropec J, Bajpeyi S, Koza RA, Rogers RC, Cefalu WT, Mynatt RL, Kozak LP. (2011) Inactivation of the mitochondrial carrier SLC25A25 (ATP-Mg2+/Pi transporter) reduces physical endurance and metabolic efficiency in mice. J. Biol. Chem. 286:11659-11671. Aprille JR (1993) Mechanism and regulation of the mitochondrial ATP-Mg/P(i) carrier. J. Bioenerg. Biomembr. 25:473-481.

TE

D

Aprille JR, Nosek MT, Brennan WA, Jr. (1982) Adenine nucleotide content of liver mitochondria increases after glucagon treatment of rats or isolated hepatocytes. Biochem. Biophys. Res. Commun. 108:834-839.

CE P

Bernardi P, Rasola A, Forte M, Lippe G. (2015) The Mitochondrial Permeability Transition Pore: Channel Formation by F-ATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology. Physiol. Rev. 95:1111-1155.

AC

Cavero S., Vozza A., del Arco A., Palmieri L., Villa A., Blanco E., Runswick M. J., Walker J. E., Cerdán S., Palmieri F., Satrústegui J. (2003) Identification and metabolic role of the mitochondrial aspartate–glutamate transporter in Saccharomyces cerevisiae. Mol. Microbiol. 50:1257–1269. Cavero S, Traba J, Del Arco A, Satrústegui J. (2005) The calcium-dependent ATP-Mg/Pi mitochondrial carrier is a target of glucose-induced calcium signalling in Saccharomyces cerevisiae. Biochem. J. 392:537-544. Chen XJ. (2004) Sal1p, a calcium-dependent carrier protein that suppresses an essential cellular function associated With the Aac2 isoform of ADP/ATP translocase in Saccharomyces cerevisiae. Genetics 167:607-617. Contreras L, Gomez-Puertas P, Iijima M, Kobayashi K, Saheki T, Satrústegui J. (2007) Ca2+ Activation kinetics of the two aspartate-glutamate mitochondrial carriers, aralar and citrin: role in the heart malate-aspartate NADH shuttle. J. Biol. Chem. 282:7098-7106. Contreras L, Satrustegui J. (2009) Calcium signaling in brain mitochondria: interplay of malate aspartate NADH shuttle and calcium uniporter/mitochondrial dehydrogenase pathways. J. Biol. Chem. 284:7091-7099 Contreras L, Urbieta A, Kobayashi K, Saheki T, Satrústegui J. (2010) Low levels of citrin (SLC25A13) expression in adult mouse brain restricted to neuronal clusters. J. Neurosci. Res. 88:1009-1016. del Arco A, Satrústegui J. (1998) Molecular cloning of Aralar, a new member of the mitochondrial carrier superfamily that binds calcium and is present in human muscle and brain. J. Biol. Chem. 273:23327-23334.

16

ACCEPTED MANUSCRIPT del Arco A, Agudo M, Satrústegui J. (2000) Characterization of a second member of the subfamily of calciumbinding mitochondrial carriers expressed in human non-excitable tissues. Biochem. J. 345:725-732.

T

del Arco A, Morcillo J, Martínez-Morales JR, Galián C, Martos V, Bovolenta P, Satrústegui J. (2002) Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues. Eur. J. Biochem. 269:3313-3320.

IP

del Arco A, Satrústegui J. (2004) Identification of a Novel Human Subfamily of Mitochondrial Carriers with Calcium-binding Domains. J. Biol. Chem. 279:24701-24713.

SC R

del Arco A, Satrústegui J. (2005) New mitochondrial carriers: an overview. Cell. Mol. Life Sci. 62: 2204-2227. del Arco A. (2005) Novel variants of human SCaMC-3, an isoform of the ATP-Mg/P(i) mitochondrial carrier, generated by alternative splicing from 3'-flanking transposable elements. Biochem. J. 389:647-655.

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Dimmock D, Maranda B, Dionisi-Vici C, Wang J, Kleppe S, Fiermonte G, Bai R, Hainline B, Hamosh A, O'Brien WE, Scaglia F, Wong LJ. (2009) Citrin deficiency, a perplexing global disorder. Mol. Genet. Metab. 96:44-9.

MA

Du J, Cleghorn W, Contreras L, Linton JD, Chan GC, Chertov AO, Saheki T, Govindaraju V, Sadilek M, Satrústegui J, Hurley JB. (2013) Cytosolic reducing power preserves glutamate in retina. Proc. Natl. Acad. Sci. U. S. A. 110:18501-18506.

TE

D

Falk MJ, Li D, Gai X, McCormick E, Place E, Lasorsa FM, Otieno FG, Hou C, Kim CE, Abdel-Magid N, Vazquez L, Mentch FD, Chiavacci R, Liang J, Liu X, Jiang H, Giannuzzi G, Marsh ED, Yiran G, Tian L, Palmieri F, Hakonarson H. (2014) AGC1 Deficiency Causes Infantile Epilepsy, Abnormal Myelination, and Reduced N-Acetylaspartate. JIMD Rep. 14:77-85.

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Fiermonte G, Palmieri L, Todisco S, Agrimi G, Palmieri F, Walker JE. (2002) Identification of the mitochondrial glutamate transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms. J. Biol. Chem. 277:19289-19294.

AC

Fiermonte G, De Leonardis F, Todisco S, Palmieri L, Lasorsa FM, Palmieri F. (2004) Identification of the Mitochondrial ATP-Mg/Pi Transporter: Bacterial expression, reconstitution, functional characterization and tissue distribution. J. Biol. Chem. 279:30722-30730. Fu HY, Zhang SR, Wang XH, Saheki T, Kobayashi K, Wang JS. (2011) The mutation spectrum of the SLC25A13 gene in Chinese infants with intrahepatic cholestasis and aminoacidemia. J. Gastroenterol. 46:510-518. Gellerich FN, Gizatullina Z, Arandarcikaite O, Jerzembek D, Vielhaber S, Seppet E, Striggow F. (2009) Extramitochondrial Ca2+ in the nanomolar range regulates glutamate-dependent oxidative phosphorylation on demand. PLoS One 4:e8181. Gellerich FN, Gizatullina Z, Trumbekaite S, Korzeniewski B, Gaynutdinov T, Seppet E, Vielhaber S, Heinze HJ, Striggow F. (2012) Cytosolic Ca2+ regulates the energization of isolated brain mitochondria by formation of pyruvate through the malate-aspartate shuttle. Biochem. J. 443:747-55 Gellerich FN, Gizatullina Z, Gainutdinov T, Muth K, Seppet E, Orynbayeva Z, Vielhaber S.(2013) The control of brain mitochondrial energization by cytosolic calcium: the mitochondrial gas pedal. IUBMB Life. 65:180-90. Giacomello M, Drago I, Bortolozzi M, Scorzeto M, Gianelle A, Pizzo P, Pozzan T. (2010) Ca2+ hot spots on the mitochondrial surface are generated by Ca2+ mobilization from stores, but not by activation of store-operated Ca2+ channels. Mol. Cell. 38:280-290.

17

ACCEPTED MANUSCRIPT Gifford JL, Walsh MP, Vogel HJ. (2007) Structures and metal-ion-binding properties of the Ca2+-binding helixloop-helix EF-hand motifs. Biochem. J. 405:199-221. Glancy B, Balaban RS (2012) Role of mitochondrial Ca2+ in the regulation of cellular energetics. Biochemistry 51:2959-2973.

IP

T

Gómez-Galán M, Makarova J, Llorente-Folch I, Saheki T, Pardo B, Satrústegui J, Herreras O. (2012) Altered postnatal development of cortico-hippocampal neuronal electric activity in mice deficient for the mitochondrial aspartate-glutamate transporter. J. Cereb. Blood Flow Metab. 32:306-317.

SC R

Harborne SP, Ruprecht JJ, Kunji ER. (2015) Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier. Biochim. Biophys. Acta 1847:1245-53.

NU

Haynes RC Jr, Picking RA, Zaks WJ. (1986) Control of mitochondrial content of adenine nucleotides by submicromolar calcium concentrations and its relationship to hormonal effects. J. Biol. Chem. 261:1612116125.

MA

Hoeflich KP, Ikura M. (2002) Calmodulin in action: diversity in target recognition and activation mechanisms. Cell 108:739-742.

D

Hoffman NE, Chandramoorthy HC, Shanmughapriya S, Zhang XQ, Vallem S, Doonan PJ, Malliankaraman K, Guo S, Rajan S, Elrod JW, Koch WJ, Cheung JY, Madesh M. (2014) SLC25A23 augments mitochondrial Ca²⁺ uptake, interacts with MCU, and induces oxidative stress-mediated cell death. Mol. Biol. Cell. 25:936-947.

CE P

TE

Irimia M, Weatheritt RJ, Ellis JD, Parikshak NN, Gonatopoulos-Pournatzis T, Babor M, Quesnel-Vallières M, Tapial J, Raj B, O'Hanlon D, Barrios-Rodiles M, Sternberg MJ, Cordes SP, Roth FP, Wrana JL, Geschwind DH, Blencowe BJ. (2014) A highly conserved program of neuronal microexons is misregulated in autistic brains. Cell 159:1511-1523.

AC

Jalil MA, Begum L, Contreras L, Pardo B, Iijima M, Li MX, Ramos M, Marmol P, Horiuchi M, Shimotsu K, Nakagawa S, Okubo A, Sameshima M, Isashiki Y, Del Arco A, Kobayashi K, Satrústegui J, Saheki T. (2005) Reduced N-acetylaspartate levels in mice lacking aralar, a brain- and muscle-type mitochondrial aspartateglutamate carrier. J. Biol. Chem. 280:31333-31339. Joyal JL, Aprille JR. (1992) The ATP-Mg/Pi carrier of rat liver mitochondria catalyzes a divalent electroneutral exchange. J. Biol. Chem. 267:19198-203. Kirichok Y, Krapivinsky G, Clapham DE. (2004) The mitochondrial calcium uniporter is a highly selective ion channel Nature 2004 427:360-364. Kobayashi K, Sinasac DS, Iijima M, Boright AP, Begum L, Lee JR, Yasuda T, Ikeda S, Hirano R, Terazono H, Crackower MA, Kondo I, Tsui LC, Scherer SW, Saheki T. (1999) The gene mutated in adult-onset type II citrullinaemia encodes a putative mitochondrial carrier protein. Nat. Genet. 22: 159-63. Korzeniewski B. (2007) Regulation of oxidative phosphorylation through parallel activation. Biophys Chem. 129:93-110. Kristián T, Pivovarova NB, Fiskum G, Andrews SB. (2007) Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention. J. Neurochem. 102:1346–1356. Kunji ER. (2004) The role and structure of mitochondrial carriers. FEBS Lett. 564:239-244. Kunji ER, Crichton PG. (2010) Mitochondrial carriers function as monomers. Biochim. Biophys. Acta 1797:817731.

18

ACCEPTED MANUSCRIPT LaNoue KF, Tischler ME. (1974) Electrogenic characteristics of the mitochondrial glutamate-aspartate antiporter. J. Biol. Chem. 249:7522-7528.

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Lasorsa FM, Pinton P, Palmieri L, Fiermonte G, Rizzuto R, Palmieri F. (2003) Recombinant expression of the Ca(2+)-sensitive aspartate/glutamate carrier increases mitochondrial ATP production in agonist-stimulated Chinese hamster ovary cells. J. Bio.l Chem. 278:38686-38692.

SC R

IP

Leverve XM, Verhoeven AJ, Groen AK, Meijer AJ, Tager JM. (1986) The malate/aspartate shuttle and pyruvate kinase as targets involved in the stimulation of gluconeogenesis by phenylephrine. Eur. J. Biochem. 155:551556. Li YI, Sánchez-Pulido L, Haerty W, Ponting CP. (2015) RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts. Genome Res. 25:1-13.

NU

Lindsay KJ, Du J, Sloat SR, Contreras L, Linton JD, Turner SJ, Sadilek M, Satrústegui J, Hurley JB. (2014) Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina. Proc. Natl. Acad. Sci. U. S. A. 111:15579-15584.

MA

Llorente-Folch I, Rueda CB, Amigo I, del Arco A, Saheki T, Pardo B, Satrustegui J. (2013) Calcium-Regulation of Mitochondrial Respiration Maintains ATP Homeostasis and Requires ARALAR/AGC1-Malate Aspartate Shuttle in Intact Cortical Neurons. J. Neurosci. 33:13957-13971.

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Llorente-Folch I, Rueda CB, Pardo B, Szabadkai G, Duchen MR, Satrustegui J. (2015) The regulation of neuronal mitochondrial metabolism by calcium. J. Physiol. 593:3447-3462.

CE P

TE

Lorenz A, Lorenz M, Vothknecht UC, Niopek-Witz S, Neuhaus HE, Haferkamp I. (2015) In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca(2+)-effects. BMC Plant Biol. 15:238. Maier H, Wang-Eckhardt L, Hartmann D, Gieselmann V, Eckhardt M. (2015) N-Acetylaspartate Synthase Deficiency Corrects the Myelin Phenotype in a Canavan Disease Mouse Model But Does Not Affect Survival Time. J. Neurosci. 35:14501-14516.

AC

Mármol P, Pardo B, Wiederkehr A, del Arco A, Wollheim CB, Satrústegui J. (2009) Requirement for aralar and its Ca2+-binding sites in Ca2+ signal transduction in mitochondria from INS-1 clonal beta-cells. J. Biol. Chem. 284:515-524. Mashima H, Ueda N, Ohno H, Suzuki J, Ohnishi H, Yasuda H, Tsuchida T, Kanamaru C, Makita N, Iiri T, Omata M, Kojima I. (2003) A novel mitochondrial Ca2+-dependent solute carrier in the liver identified by mRNA differential display. J. Biol. Chem. 278:9520-9527. McCormack, J.G., Halestrap, A.P. Denton, R.M. (1990) Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiol. Rev. 70:391-425. Monné M, Miniero DV, Obata T, Daddabbo L, Palmieri L, Vozza A, Nicolardi MC, Fernie AR, Palmieri F. (2015) Functional characterization and organ distribution of three mitochondrial ATP-Mg/Pi carriers in Arabidopsis thaliana. Biochim. Biophys. Acta 1847:1220-1230. Napolioni V, Persico AM, Porcelli V, Palmieri L. (2011) The mitochondrial aspartate/glutamate carrier AGC1 and calcium homeostasis: physiological links and abnormalities in autism. Mol. Neurobiol. 44:83-92. Nicholls DG, Chalmers S. (2004) The integration of mitochondrial calcium transport and storage. J Bioenerg Biomembr. 36:277–281.

19

ACCEPTED MANUSCRIPT Nosek MT, Dransfield DT, Aprille JR (1990) Calcium stimulates ATP-Mg/Pi carrier activity in Rat liver mitochondria. J. Biol. Chem. 265: 8444-8450. Nury H, Dahout-Gonzalez C, Trézéguet V, Lauquin GJ, Brandolin G, Pebay-Peyroula E. (2006) Relations between structure and function of the mitochondrial ADP/ATP carrier. Annu. Rev. Biochem. 75:713-741.

IP

T

Palmieri L, Pardo B, Lasorsa FM, del Arco A, Kobayashi K, Iijima M, Runswick MJ, Walker JE, Saheki T, Satrústegui J, Palmieri F. (2001) Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria. EMBO J. 20:5060-5069.

SC R

Palmieri F. (2004) The mitochondrial transporter family (SLC25): physiological and pathological implications. Pflugers Arch. 447:689-709.

NU

Palmieri F. (2013) The mitochondrial transporter family SLC25: identification, properties and physiopathology. Mol. Asp. Med. 34:465-484. Palmieri F, Monné M. (2016) Discoveries, metabolic roles and diseases of mitochondrial carriers: A review. Biochim Biophys Acta doi: 10.1016/j.bbamcr.2016.03.007.

MA

Pardo B, Contreras L, Serrano A, Ramos M, Kobayashi K, Iijima M, Saheki T, Satrústegui J. (2006) Essential role of aralar in the transduction of small Ca2+ signals to neuronal mitochondria. J. Biol. Chem. 281:1039-1047.

TE

D

Pardo B, Rodrigues TB, Contreras L, Garzón M, Llorente-Folch I, Kobayashi K, Saheki T, Cerdan S, Satrústegui J. (2011) Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation. J. Cereb. Blood Flow Metab. 31:90-101.

CE P

Pebay-Peyroula E, Dahout-Gonzalez C, Kahn R, Trézéguet V, Lauquin GJ, Brandolin G. (2003) Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature 426:39-44. Ramos M, del Arco A, Pardo B, Martínez-Serrano A, Martínez-Morales JR, Kobayashi K, Yasuda T, Bogónez E, Bovolenta P, Saheki T, Satrústegui J. (2003) Developmental changes in the Ca2+-regulated mitochondrial aspartate-glutamate carrier aralar1 in brain and prominent expression in the spinal cord. Brain Res Dev Brain Res. 143:33-46.

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Ramos M, del Arco A, Pardo B, Martínez-Serrano A, Martínez-Morales JR, Kobayashi K, Yasuda T, Bogónez E, Bovolenta P, Saheki T, Satrústegui J.Ramos M, Pardo B, Llorente-Folch I, Saheki T, del Arco A, Satrústegui J. (2011) Deficiency of the mitochondrial transporter of aspartate/glutamate aralar/AGC1 causes hypomyelination and neuronal defects unrelated to myelin deficits in mouse brain. J Neurosci Res. 89:20082017. Robinson AJ, Kunji ER. (2006) Mitochondrial carriers in the cytoplasmic state have a common substrate binding site. Proc. Natl. Acad. Sci. U. S. A. 103: 2617-2622. Robinson AJ, Overy C, Kunji ERS. (2008) The mechanism of transport by mitochondrial carriers based on analysis of symmetry. Proc. Natl. Acad. Sci. U. S. A. 105: 17766-17771. Rubi B, del Arco A, Bartley C, Satrustegui J, Maechler P. (2004) The malate-aspartate NADH shuttle member Aralar1 determines glucose metabolic fate, mitochondrial activity, and insulin secretion in beta cells. J. Biol. Chem. 279:55659-55666. Rueda CB, Llorente-Folch I, Amigo I, Contreras L, González-Sánchez P, Martínez-Valero P, Juaristi I, Pardo B, del Arco A, Satrústegui J. (2014) Ca(2+) regulation of mitochondrial function in neurons. Biochim. Biophys. Acta 1837:1617-1624.

20

ACCEPTED MANUSCRIPT Rueda CB, Traba J, Amigo I, Llorente-Folch I, González-Sánchez P, Pardo B, Esteban JA, del Arco A, Satrústegui J. (2015) Mitochondrial ATP-Mg/Pi carrier SCaMC-3/Slc25a23 counteracts PARP-1-dependent fall in mitochondrial ATP caused by excitotoxic insults in neurons. J. Neurosci. 35:3566-3581.

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Run C, Yang Q, Liu Z, OuYang B, Chou JJ (2015) Molecular Basis of MgATP Selectivity of the Mitochondrial SCaMC Carrier. Structure 23:1394-403.

SC R

IP

Saheki T and Kobayashi K (2002) Mitochondrial aspartate glutamate carrier (citrin) deficiency as the cause of adult-onset type II citrullinemia (CTLN2) and idiopathic neonatal hepatitis (NICCD). Journal of Human Genetics 47: 333-41.

NU

Saheki T, Iijima M, Li MX, Kobayashi K, Horiuchi M, Ushikai M, Okumura F, Meng XJ, Inoue I, Tajima A, Moriyama M, Eto K, Kadowaki T, Sinasac DS, Tsui LC, Tsuji M, Okano A, Kobayashi T. (2007) Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency. J. Biol .Chem. 282:25041-25052.

MA

Saheki T, Inoue K, Ono H, Tushima A, Katsura N, Yokogawa M, Yoshidumi Y, Kuhara T, Ohse M, Eto K, Kadowaki T, Sinasac DS, Kobayashi K. (2011) Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency. Mol. Genet. Metab. 104:492-500.

TE

D

Sakurai T, Ramoz N, Barreto M, Gazdoiu M, Takahashi N, Gertner M, Dorr N, Gama Sosa MA, De Gasperi R, Perez G, Schmeidler J, Mitropoulou V, Le HC, Lupu M, Hof PR, Elder GA, Buxbaum JD. (2010) Slc25a12 disruption alters myelination and neurofilaments: a model for a hypomyelination syndrome and childhood neurodevelopmental disorders. Biol Psychiatry 67:887-94.

CE P

Saraste M, Walker JE. (1982) Internal sequence repeats and the path of polypeptide in mitochondrial ADP/ATP translocase. FEBS Lett. 144:250-254. Satrústegui J, Pardo B. del Arco A (2007a) Mitochondrial transporters as novel targets for intracellular calcium signaling. Physiol. Rev. 87:29-67.

AC

Satrústegui J, Contreras L, Ramos M, Mármol P, del Arco A, Saheki T, Pardo B. (2007b) Role of aralar, the mitochondrial transporter of aspartate-glutamate, in brain N-acetylaspartate formation and Ca(2+) signaling in neuronal mitochondria. J. Neurosci. Res. 85: 3359-3366. Satrústegui J, Bak LK. (2015) Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle: Implications for Neuronal Energy Metabolism. Neurochem. Res. 40:2425-2430. Song YZ, Zhang ZH, Lin WX, Zhao XJ, Deng M, Ma YL, Guo L, Chen FP, Long XL, He XL, Sunada Y, Soneda S, Nakatomi A, Dateki S, Ngu LH, Kobayashi K, Saheki T. (2013) SLC25A13 gene analysis in citrin deficiency: sixteen novel mutations in East Asian patients, and the mutation distribution in a large pediatric cohort in China. PLoS One 8:e74544. Stael S, Rocha AG, Robinson AJ, Kmiecik P, Vothknecht UC, Teige M. (2011) Arabidopsis calcium-binding mitochondrial carrier proteins as potential facilitators of mitochondrial ATP-import and plastid SAM-import. FEBS Lett. 585:3935-3940. Thangaratnarajah C, Ruprecht JJ, Kunji ER. (2014) Calcium-induced conformational changes of the regulatory domain of human mitochondrial aspartate/glutamate carriers. Nat. Commun. 5:5491.

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ACCEPTED MANUSCRIPT Traba J, Froschauer EM, Wiesenberger G, Satrústegui J, del Arco A. (2008) Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose. Mol. Microbiol. 69:570-585.

T

Traba J, Satrústegui J, del Arco A. (2009) Characterization of SCaMC-3-like/slc25a41, a novel calciumindependent mitochondrial ATP-Mg/Pi carrier. Biochem. J. 418: 125-133.

SC R

IP

Traba J, del Arco A, Duchen MR, Szabadkai G, Satrústegui J. (2012) SCaMC-1 promotes cancer cell survival by desensitizing mitochondrial permeability transition via ATP/ADP-mediated matrix Ca2+ buffering. Cell Death Differ. 19:650-660. Urano T, Shiraki M, Sasaki N, Ouchi Y, Inoue S. (2015) SLC25A24 as a novel susceptibility gene for low fat mass in humans and mice. J. Clin. Endocrinol. Metab. 100:E655-63.

NU

Weber FE, Minestrini G, Dyer JH, Werder M, Boffelli D, Compassi S, Wehrli E, Thomas RM, Schulthess G, Hauser H. (1997) Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily. Proc Natl Acad Sci U S A. 94:8509-8514.

MA

Wibom R, Lasorsa FM, Töhönen V, Barbaro M, Sterky FH, Kucinski T, Naess K, Jonsson M, Pierri CL, Palmieri F, Wedell A. (2009) AGC1 deficiency associated with global cerebral hypomyelination. N. Engl. J. Med. 361:489495.

TE

D

Wongkittichote P, Tungpradabkul S, Wattanasirichaigoon D, Jensen LT. (2013) Prediction of the functional effect of novel SLC25A13 variants using a S. cerevisiae model of AGC2 deficiency. J. Inherit. Metab Dis. 36:82130.

AC

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Yang Q, Brüschweiler S, Chou JJ. (2014) A self-sequestered calmodulin-like Ca²⁺ sensor of mitochondrial SCaMC carrier and its implication to Ca²⁺-dependent ATP-Mg/P(i) transport. Structure 22:209-217.

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Table 1. Summary of prominent features of the regulatory N-domains of CaMCs N-domain features Length and number of EF-hands

References

Ca2+ -binding* Half-maximal activation by Ca2+

Functional approach used

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CaMC protein

Calcium activation

330 residues 8 EF-hands (EF-4/EF-8 non-functional)

Yes

330 residues 8 EF-hands (EF-4/EF-8 non-functional)

Yes

142 ± 38 nM

nd

3.3 ± 0.9 μM

 300 nM

SCaMC-1/SLC25A24

168 residues 4 EF-hands

Yes

SCaMC-2a/SLC25A25

160 residues 3 EF-hands

Yes

206 residues 4 EF-hands (EF1 and EF4 non-functional)

A. thaliana AtAPC1 At5g61810

182 residues 4 EF-hands

A. thaliana AtAPC2 (At5g51050) A. thaliana AtAPC3 (At5g07320)

Yes

15.0 ± 1.1 μM

 0.2 μM

186 residues 4 EF-hands

Yes

 0.2 μM

183 residues 4 EF-hands)

Yes

 0.8 μM

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Yes

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del Arco and Satrustegui, 1998 Pardo et al., 2006 Gellerich et al., 2009

Ca2+-activation of MAS activity in isolated mouse liver mitochondria

del Arco et al., 2000 Contreras et al., 2007

ATP uptake in isolated mouse liver mitochondria

Amigo et al., 2013

2+

Ca -activation of carboxyatractylosideinsensitive ATP transport in isolated mitochondria from a COS-7 cell line expressing mitochondrial luciferase

Weber et al., 1997 Traba et al., 2012 Mashima et al., 2003

nd

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S. cerevisiae Sal1p (YNL083W)

12.7 ± 5.3  μM

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159 residues 4 EF-hands

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ATP-Mg/Pi carriers SCaMC-3/SLC25A23

Ca2+ activation of Malate/Aspartate Shuttle (MAS) activity in isolated rat brain mitochondria . Ca2+-activation of respiratory state 3 with glutamate plus malate

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Citrin/AGC2/ SLC25A13

324 ± 57 nM

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Aralar/AGC1/ SLC25A12

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Aspartate/glutamate carriers

Ca2+-activation of carboxyatractylosideinsensitive ATP transport in isolated yeast mitochondria expressing mitochondrial luciferase Ca2+-activation of ADP/ATP transport assay of recombinant proteins reconstituted into proteoliposomes Ca2+-activation of ADP/ATP transport assay of recombinant proteins reconstituted into proteoliposomes Ca2+-activation of ADP/ATP transport assay of recombinant proteins reconstituted into proteoliposomes

Chen et al., 2004 Traba et al., 2008 Stael et al., 2011 Monné et al 2015 Stael et al., 2011 Monné et al 2015 Stael et al., 2011 Monné et al 2015

* Ca2+-binding to the purified N-terminal domains determined by 45Ca2+-overlay assay. nd; not determined

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Figure 1

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Figure 2

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Figure 3

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