**1. Introduction**

An ancestral endosymbiotic α-proteobacteria fused with an ancestral eukaryotic cell to give rise to the mitochondria approximately 1.5 billion years ago [1]. Since then, metabolic processes involving multiple compartments of the cell are facilitated by specific transmembrane transporter proteins [2]. The intracellular transport of mitochondrial metabolites plays an important role in cellular respiration via the processes of the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, amino acids biosynthesis [3], fatty acids biosynthesis [4], photorespiration [5], and C4 photosynthesis [6]. Metabolic intermediates of these pathways pass through the double membrane of mitochondria that delineates four different sub-compartments: the outer mitochondrial membrane (OMM), the intermembrane space (IMS), the inner mitochondrial membrane (IMM), and the mitochondrial matrix (MM) [7]. The OMM is highly permissive to the passage of ions and small uncharged molecules (<5 kDa) through pore-forming membrane proteins (porins), such as the voltage-dependent anion channels [8]. Larger molecules, especially proteins, must be imported by specialized translocases. By contrast, the IMM is a more stringent molecular barrier allowing only specific metabolites to cross from or into the MM [9]. The highly impermeable IMM is required to establish an electrochemical

gradient via the activities of the oxidative phosphorylation membrane protein complexes needed for ATP biosynthesis [10]. Thus, an array of nuclear-encoded mitochondrial carrier (MC) proteins are responsible for the transport of a wide range of substrates shuttled across the IMM. MCs belong to a superfamily of transporters called the mitochondrial carrier family (MCF). Various aspects of plant MCFs have been extensively reviewed by Palmieri et al. (2011) [11], Monné et al. (2019) [3], Haferkamp and Schmitz-Esser (2012) [12], and Lee and Millar (2016) [13].

Most MCF members are relatively small, ranging from 30 to 35 kDa, around 300 amino acids in length, and have a conserved six transmembrane α-helices region [14]. Most of the primary structure of MCs is comprised of three homologous regions, each approximately 100 amino acids in length [15], and both the N and C termini face the IMS [16]. Each repeat region is comprised of two transmembrane segments flanking a short helical region that is oriented parallel to the lipid bilayer [17]. Furthermore, each repeat region is comprised of two hydrophobic transmembrane α-helices connected by a long hydrophilic matrix loop [3,18] and bears the mitochondrial carrier domain superfamily motif (IPR023395). In the odd-numbered α-helices is the conserved PX[DE]XX[RK] motif, the charged residues in this motif can form inter-domain salt bridges (also called matrix salt-bridge network) [19–21]. Residues of the salt-bridge form a hydrogen bond with a proximal glutamine residue stabilizing the network (also called Q brace) [19–21]. Moreover, in the even-numbered α-helices is another conserved [YF][DE]xx[KR] motif that can form another salt bridge called the cytoplasmic salt-bridge network [19–21]. This network is stabilized by the Y brace, hydrogen bonds formed by the tyrosine residue in the motif [19–21].

Decades of biochemical and structural data have shed light on the transport mechanism of MCs [21,22]. This transport cycle involves what is now referred to as the alternating access mechanism [20] but was previously described by a number of groups as "gated pore model" [23] or "ping-pong mechanism" [24]. The mechanism describes the process wherein a substrate binds to the transporter in its c-state (cytoplasmic-side open and matrix-side closed state), undergoes a conformational change to a transition state and to an m-state (matrix-side open and cytoplasmic-side closed state), followed by the release of the substrate into the MM [17,20,23,25–27]. The counter-substrate binds to the transporter in the m-state, undergoes a conformational change eventually leading to the c-state, and releases the counter-substrate into the cytosol [17,20,23,25–27]. Upon release of the counter-substrate, the transporter is ready to begin a new transport cycle. The c-state confirmation was confirmed by the crystal structure of the ADP/ATP carrier in complex with the inhibitor carboxyatractyloside [17]. Recently, an ADP/ATP carrier in its m-state was crystallized in complex with another inhibitor, bongkrekic acid [20]. The transport cycle mechanism of MCFs has recently been reviewed [21,28].

While ADP/ATP carriers have been studied extensively, much is yet to be learned from other members of the family, particularly among plant MCF members. In *Arabidopsis thaliana*, 58 MCF proteins have been identified, but in vitro studies indicate broad substrate specificities and their physiological function *in planta* is largely unknown [11–13]. This lack of specificity among plant MCFs is surprising given the metabolic control expected to exist at the IMM. Among the substrates that have been demonstrated to be transported by Arabidopsis MCs across the IMM are: nucleotides and dinucleotides (ATP, ADP, AMP, NAD+, FAD/folate); di-/tricarboxylates (malate, succinate, 2-oxoglutarate (2-OG), oxaloacetate (OAA), fumarate, citrate, isocitrate); amino acids (glutamate, aspartate, *S*-adenosylmethionine); cofactors (coenzyme A, thiamine diphosphate); and ions (phosphates, protons, Fe2+/3+) [3,12]. Most MC proteins are localized to the IMM, while a few are localized elsewhere. For example, AT5G17400 (ER-ANT) is targeted to the endoplasmic reticulum [29] and some members like AT4G32400 (AtBrittle1) are dual targeted to the mitochondria and plastids [30]. Of the 58 MCF members in *A. thaliana*, only 28 have thus far been confirmed to localize to the mitochondria by organellar proteomics and localization by fluorescent protein tagging, while a total of 12 MCF members have been reported to localize elsewhere [13]. Here, we focus on a subset of MCs that are potentially

involved in cellular respiration either in the transport of TCA cycle intermediates or in transport processes relevant to mitochondrial oxidative phosphorylation.
