**Proteomic and Bioinformatic Profiling of Transporters in Higher Plant Mitochondria**

#### **Ian Max Møller 1, \* , R. Shyama Prasad Rao 2 , Yuexu Jiang 3 , Jay J. Thelen <sup>4</sup> and Dong Xu 3**


Received: 9 July 2020; Accepted: 13 August 2020; Published: 16 August 2020

**Abstract:** To function as a metabolic hub, plant mitochondria have to exchange a wide variety of metabolic intermediates as well as inorganic ions with the cytosol. As identified by proteomic profiling or as predicted by MU-LOC, a newly developed bioinformatics tool, *Arabidopsis thaliana* mitochondria contain 128 or 143 different transporters, respectively. The largest group is the mitochondrial carrier family, which consists of symporters and antiporters catalyzing secondary active transport of organic acids, amino acids, and nucleotides across the inner mitochondrial membrane. An impressive 97% (58 out of 60) of all the known mitochondrial carrier family members in *Arabidopsis* have been experimentally identified in isolated mitochondria. In addition to many other secondary transporters, *Arabidopsis* mitochondria contain the ATP synthase transporters, the mitochondria protein translocase complexes (responsible for protein uptake across the outer and inner membrane), ATP-binding cassette (ABC) transporters, and a number of transporters and channels responsible for allowing water and inorganic ions to move across the inner membrane driven by their transmembrane electrochemical gradient. A few mitochondrial transporters are tissue-specific, development-specific, or stress-response specific, but this is a relatively unexplored area in proteomics that merits much more attention.

**Keywords:** ABC transporter; aquaporin; ATP synthase; ion channels; mitochondrial carrier family

#### **1. Introduction**

Eukaryotic multicellular organisms need to exchange energy, matter, and information between the environment and their cells, between their cells, and within their cells. To perform these tasks, they need a diverse array of specialized proteins to move ions and molecules across the biological membranes, which delimit the cells and the subcellular compartmentation. These proteins are collectively known as transporters, which include carriers, channels, and pumps [1].

The mitochondrion is a metabolic hub, not only for energy metabolism—the tricarboxylic acid (TCA) cycle and oxidative phosphorylation—but also for the biosynthesis of coenzymes, amino acids, some fatty acids, and lipids [2,3]. In photosynthetic cells in the light, there is a massive flow of fixed carbon from the chloroplasts to the rest of the cell, especially to the mitochondria via the cytosol, but metabolic cooperation between plastids and mitochondria also takes place in darkness [4,5]. Retrograde signaling from the mitochondria to the nucleus probably involves export of peptides [6,7]. In addition to this, to grow and divide the mitochondria one needs to import the vast majority of its proteins as well as some tRNAs and rRNA [8–10].

All of these processes require the presence of many different transporters in the mitochondria. The outer membrane contains only two types: (i) Porin also called voltage-dependent anion channel (VDAC) or voltage-dependent gated ion channel (VIC), which makes the outer mitochondrial membrane (OMM) permeable to all molecules smaller than 5 kDa, obviating the need for other transporters of small ions and molecules [11,12]. (ii) Translocase Outer Membrane (TOM), the subcomplex of the Mitochondrial Protein Translocase (MPT) responsible for importing proteins across the OMM. In addition to Translocase Inner Membrane (TIM), the MPT subcomplex responsible for importing proteins across the inner mitochondrial membrane (IMM), the IMM contains many other transporters of several different classes. The mitochondrial transportome was comprehensively reviewed by Lee and Millar [13].

It is the purpose of this review first to compile a list of the transporters identified by proteomic profiling of isolated plant mitochondria. This list will then be compared to a list of transporters predicted by MU-LOC [14], a newly developed program, to predict mitochondrial proteins based on their amino acid sequences and their gene expression patterns. Finally, for each transporter class or family, we will briefly discuss the properties of the transporters present in plant mitochondria.

#### **2. The Experimental Proteome and Transportome in Plant Mitochondria**

#### *2.1. The Experimental Mitochondrial Proteome*

The mitochondrial proteome has been characterized in some depth in *Arabidopsis thaliana* cell cultures and in potato (*Solanum tuberosum* L.) tubers. In both, almost 1100 proteins were identified as summarized by Rao et al. [8]. Since then, Senkler et al. [15] published what they called the ''mitochondrial complexome of *Arabidopsis thaliana*", in which they identified 1359 proteins involved in various complexes both in the membranes and in the soluble fraction. Altogether, the mitochondrial proteome in a plant probably contains 2000–2500 proteins [8] or about 10% of the total *Arabidopsis* proteome (Table 1). Plant mitochondrial DNA encodes at most 40 proteins [16], which are synthesized inside the mitochondria on mitochondrial ribosomes, while the remaining 2000+ proteins are encoded in the nuclear DNA, synthesized on cytosolic ribosomes, or on polysomes associated with the mitochondrial surface, and imported across the OMM and IMM [17].

The largest protein groups in the identified proteome of both *Arabidopsis* and potato mitochondria are related to energy and metabolism, with around 150 and 200 proteins, respectively; protein fate, protein synthesis, and RNA processing are each represented by approximately 100 proteins, while transport has around 50 proteins [8]. In this Gene Ontology (GO) nomenclature, many transporters are listed under different GO terms, e.g., ATP synthase subunits are found under energy. The actual number of identified transporters is therefore much larger, as discussed below. However, it is not just the number of unique proteins that is important. The abundance of each protein is also important, and Salvato et al. [18] used spectral counting to estimate the abundance of the identified proteins. The 52 proteins under the GO term Transporter showed an overall average abundance, but with significant variation among the transporters. Fuch et al. [19] went one step further by estimating the copy number of the individual proteins and protein complexes present in a single mitochondrion. Based on that, they could then calculate the surface area occupied by the various membrane proteins. They found that VDAC and TOM cover 34% and 12% of the surface area of the OMM, respectively. The five respiratory complexes cover 18% of the IMM, while the most abundant of the other carriers—ADP/ATP carrier, phosphate carrier, the uncoupling protein, and the tricarboxylate/dicarboxylate carrier—cover a total of about 11% of the IMM [19].


**Table 1.** Number of proteins in different transporter classes and families, and their experimental (proteomic) identification in isolated mitochondria of Arabidopsis and rice and mitochondrial localization prediction status (using MU-LOC) in Arabidopsis, rice, human, and mouse.
