Update of the sideroflexin (SLC56) gene family

Abstract

The human sideroflexin (SFXN) gene family, also classified as solute carrier family 56 (SLC56), encodes a group of five mitochondrial transmembrane proteins (SFXN1–SFXN5) involved in key aspects of mitochondrial metabolism, cellular homeostasis, and development. SFXNs are highly conserved across eukaryotic species, with evolutionary the origin traced back to the earliest metazoans. Functionally, each of the five family members exhibits distinct functional specialization. Particularly, SFXN1 and SFXN3 facilitate mitochondrial serine transport, supporting one-carbon metabolism. SFXN2 and SFXN4 are implicated in mitochondrial iron regulation, heme biosynthesis, and iron–sulfur cluster assembly. SFXN5, predominantly expressed in the brain, is proposed to regulate citrate metabolism and immune cell functions. Mutations or dysregulation of SFXN genes have been linked to certain human diseases, including congenital sideroblastic anemia, oxidative phosphorylation disorders, neurodegenerative conditions, and cancers. Structurally, SFXNs share conserved transmembrane domains and key motifs critical for substrate transport, mitochondrial iron homeostasis, and overall mitochondrial function. The evolutionary trajectory of the SFXN family—from amino acid transport to functionally specialized roles in higher organisms—highlights their biological and clinical significance. Comparative studies across model organisms reveal both conserved and divergent functions, emphasizing their importance in health and disease. A comprehensive understanding of the SFXN family not only advances fundamental mitochondrial research but also opens avenues for novel therapeutic interventions.

Background

The sideroflexin (SFXN) gene family is also known as the solute carrier 56 (SLC56) gene family. SLC56 is one of 66 families included in the large SLC superfamily, which comprises a total of more than 450 members [1–3]. This diverse group of membrane transporter proteins is responsible for the movement of a wide range of solutes across cellular membranes. Substrates for SLC transporters include: more than two dozen inorganic cations and anions [e.g., H⁺, Mn²⁺, Fe³⁺, (SeO₃)²⁻, (OH)⁻, (SO₄)²⁻, (PO₄)³⁻], carboxylate and other organic anions, amino acids and oligopeptides, glucose and other sugars, bile salts, acetyl coenzyme A, essential metal nutrients, biogenic amines, vitamins, urea, neurotransmitters, fatty acids, lipids, nucleosides, ammonium, choline, and thyroid hormone [3–5].

The human SLC56 (SFXN) gene family is comprised of five mitochondrial transmembrane solute-carrier proteins, designated SFXN1 through SFXN5. SFXN genes are conserved across virtually all eukaryotes, including the earliest metazoans and yeast, reflecting their evolutionary significance [6–9]. The “sideroflexin” name was derived from research on mice with two notable traits: sideroblastic anemia and “flexed tail” [7]. This study discovered a Sfxn1 gene mutation associated with the “flexed tail” trait and proposed that loss of Sfxn1 activity caused the observed sideroblastic anemia phenotype. However, this association remains unclear and has been questioned by subsequent studies [10, 11].

SFXN proteins participates in cellular metabolism, mitochondrial homeostasis, and development [7, 12]. They were initially identified in rodents as candidates for mitochondrial citrate transport [6] and have since been implicated in the transport of amino acids and other metabolites, as well as in iron homeostasis, and energy production [12–14]. Among these processes, SFXN1 functions as a mitochondrial serine transporter, which is crucial for one-carbon metabolism and associated with nucleotide biosynthesis [13, 15]. In contrast, SFXN4 lacks serine transport capability [13], but instead contributes to mitochondrial iron utilization [16]. By supporting the biogenesis of iron-sulfur clusters (ISCs) and hemoglobin synthesis, SFXN4 contributes to oxidative phosphorylation by acting as an assembly factor for complex I of the mitochondrial respiratory chain [17]. This divergence in functional roles of the various SFXNs highlights the specialized contributions of SFXN family members to cellular and metabolic processes. Mutations in, or dysregulation of, SFXN genes have been associated with severe anemia, neurodegenerative diseases, and cancer [8, 18, 19]. The evolutionary trajectory of this family is particularly intriguing. While several members mediate mitochondrial amino acid transport, SFXN4 exhibits a distinct function focused on iron–sulfur cluster biogenesis and respiratory chain assembly, reflecting an adaptive shift as a factor within mitochondrial physiology [13, 16].

The present article explores the evolutionary history, structural characteristics, and biological functions of the SFXN gene family, highlighting their participation in health and disease. By examining their structural and functional features, their importance in mitochondrial biology and human health is emphasized.

Evolution of the SFXN gene family

The sideroflexin gene family encodes a group of SFXN proteins that are highly conserved across eukaryotes, underscoring their important roles in cellular and mitochondrial function. The evolutionary path of this family offers valuable insights into how the genes have adapted to support processes in diverse organisms [4, 13]. Though bacteria lack SFXNs; they possess analogous proteins that perform similar functions in nutrient transport and metabolism. Bacterial systems — such as amino acid transporters from the Major Facilitator Superfamily (MFS) and ABC transporters, coupled with iron acquisition mechanisms (e.g., FeoB and siderophores) fulfill comparable biochemical functions in prokaryotic cells [20–22]. Homologs of SFXN genes are found across virtually all eukaryotic lineages, including plants (Physcomitrium patens, Selaginella moellendorffii), opisthokonts, (e.g., choanoflagellates), yeast (Saccharomyces cerevisiae), fruit fly (Drosophila melanogaster) and higher animals (Table 1; Fig. 1) [4, 7, 12, 13, 23]. The presence of SFXN-like proteins in simple organisms, such as fungi [24], suggests the gene family arose early in eukaryotic evolution, consistent with the emergence of mitochondria as an autonomous organelle [12, 13].

Table 1.

SFXN genes from representative species. These data were retrieved from the NCBI gene Entrez database for each species (as of February 10, 2025)

Organism	Number of SFXN genes	Gene names	Gene IDs
Homo sapiens (Human)	5	SFXN1, SFXN2, SFXN3, SFXN4, SFXN5	94,081, 118,980, 81,855, 119,559, 94,097
Mus musculus (Mouse)	5	Sfxn1, Sfxn2, Sfxn3, Sfxn4, Sfxn5	14,057, 94,279, 94,280, 94,281, 94,282
Danio rerio (Zebrafish)	6	Sfxn1, Sfxn2, Sfxn3, Sfxn4, Sfxn5a, Sfxn5b	445,143, 334,757, 791,182, 556,121, 562,654, 561,995
Xenopus tropicalis (Tropical clawed frog)	5	Sfxn1, Sfxn2, Sfxn3, Sfxn4, Sfxn5	548,998, 496,514, 100,216,274, 100,487,071, 448,290
Drosophila melanogaster (Fruit fly)	2	Sfxn1/3, Sfxn2	40,552, 40,080
Centruroides sculpturatus (Bark scorpion)	2	Sfxn1-3, Sfxn2	111,628,057, 111,639,308
Caenorhabditis elegans (Roundworm)	7	sfxn1.1, sfxn1.2, sfxn1.3, sfxn1.4, sfxn1.5, sfxn2, sfxn5	181,807, 174,708, 180,055, 183,547, 181,353, 181,054, 174,303
Rhopilema esculentum (Jellyfish)	3	Sideroflexin-1-like, sideroflexin-2-like, sideroflexin-5-like	135,695,859, 135,696,064, 135,695,829
Magallana gigas (Pacific oyster)	3	Sfxn1, Sfxn2, Sfxn5	105,326,336, 105,335,554, 105,318,942
Lytechinus variegatus (Green sea urchin)	3	Sideroflexin-1-like, sideroflexin-2-like, sideroflexin-5-like	121,424,831, 121,425,488, 121,424,772
Amphimedon queenslandica (Sponge)	3	Sideroflexin-1-like, sideroflexin-2-like, sideroflexin-5-like	100,633,500, 100,637,156, 100,637,152
Salpingoeca rosetta (Choanoflagellate)	2	Sfxn1, Sfxn5	16,074,064, 16,071,733
Selaginella moellendorffii (Lycophyte plant)	1	Sfxn5	9,632,309
Saccharomyces cerevisiae S288C (Yeast)	1	FSF1	854,445

Fig. 1.

Phylogenetic analysis of the SFXNs from representative species. The evolutionary history was inferred by using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model [61]. The tree with the highest log likelihood (-21706.14) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and Biological Neighbor-Joining (BioNJ) algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. A distance scale of 0.50 indicates 0.5 substitutions per site. This analysis involved 48 amino acid sequences. There was a total of 429 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [60]. Abbreviations: Aqu: Amphimedon queenslandica, Cel: Caenorhabditis elegans, Csc: Centruroides sculpturatus, Dme: Drosophila melanogaster, Dre: Danio rerio, Hsa: Homo sapiens, Lva: Lytechinus variegatus, Mgi: Magallana giga, Mmu: Mus musculus, Res: Rhopilema esculentum, Sce: Saccharomyces cerevisiae S288C, Smo: Selaginella moellendorffii, Sro: Salpingoeca rosetta, Xtr: Xenopus tropicalis

Although most studies have focused on animals and fungi, BLASTp results show that sideroflexin homologs exist also in plants and protists. Notably, the bryophyte Physcomitrium patens and the lycophyte Selaginella moellendorffii each contain a single SFXN gene, (sideroflexin-like and Sfxn5 respectively) (Table 1). However, no annotated SFXN genes have been found in any angiosperm or gymnosperm genome examined to date, including well-curated model species such as Arabidopsis thaliana, rice, maize and poplar.

The currently reported plant sideroflexins share only 22–37% amino acid sequence similarity with human SFXNs, with the highest similarity being to human SFXN5 (Fig. 2). This pattern suggests that sideroflexins may have been present in the earliest eukaryotes but underwent differential retention and expansion in distinct evolutionary lineages [25]. In plants, for example, iron regulation primarily relies on other mechanisms (such as phytosiderophores and citrate-based mechanisms) rather than sideroflexins [26]. In fact, there is no evidence that plants have mitochondrial serine transporters analogous to SFXN1. In plants, however, the reaction catalyzed by the mitochondrial serine hydroxymethyltransferase (SHMT) runs in the direction producing serine from glycine [27]. Thus, this reaction apparently provides mitochondrial serine in the absence of the shuttle of serine from cytosol. A choanoflagellate (e.g., Salpingoeca rosetta in Table 1) is among the opisthokonts — considered the closest living relatives of animals and the earliest functional eukaryotic unicellular organisms that have a diploid genome within the nucleus and mitochondria in the cytoplasm [28] — possesses Sfxn1 and Sfxn5. This finding indicates that SFXN genes were already established before the emergence of multicellular animals. Presence of multiple SFXN genes in the roundworm Caenorhabditis elegans (seven genes) and zebrafish Danio rerio (six genes) reflects lineage-specific expansions and possible functional diversification [12, 18].

Fig. 2.

Amino acid and peptide sequence and alignment of human and plant SFXNs. Dark purple (fully shaded regions) denotes amino acids that are identical across all sequences, while lighter purple (or partially shaded regions) indicates semi-conserved or conserved substitutions. The HPDT motif and the asparagine (N)-rich sequences are also highlighted. Unmarked residues represent sequence variations. Dashes (-) represent gaps in the alignment; these gaps suggest evolutionary divergence, i.e., where one sequence has additional or missing amino acids. The sequence numbering on the right side indicates the position of amino acids in the full-length protein sequence. Sequences were retrieved from the NCBI Gene Entrez database and aligned using Clustal Omega v1.2.4 (default parameters) via the UniProtKB/Swiss-Prot alignment tool [59]. Abbreviations: Hsa: Homo sapiens, Smo: Selaginella moellendorffii, Ppa: Physcomitrium patens. The figure was created using Biorender (https://BioRender.com)

Evolution of the SFXN gene family appears to parallel the growing complexity of mitochondrial functions in multicellular organisms. The role of SFXN family members in biological processes, such as one-carbon metabolism and iron-sulfur cluster (ISC) biosynthesis, likely emerged to meet the growing metabolic demands of larger, more complex organisms [7, 12, 24, 25]. Comparative studies in model organisms, including yeast and zebrafish, have been instrumental in identifying shared and specialized functions across species [13, 18, 29]. For example, homologous proteins in yeast, fruit fly, and vertebrates demonstrate conserved functions in mitochondrial transport, particularly in serine transport and iron homeostasis, underlining their significance in eukaryotic evolution [7, 12, 13, 23, 24].

Apparently, a SFXN5-like gene was the first to appear during evolution of the SFXN family (Figs. 1 and 3). In humans, most SFXN homologs have a high degree of sequence conservation, with SFXN1 and SFXN3 having 77% similarity. SFXN1 and SFXN2 share 55% of identical amino acid residues, while SFXN1 and SFXN4 share only 23% identical residues [12, 13] (Fig. 2). Given the potential involvement of SFXN1 and SFXN3 in mitochondrial serine transport [13], a high degree of amino acid sequence similarity is not surprising. Among mammalian SFXNs, SFXN4 is the most divergent member.

Fig. 3.

Phylogenetic comparison of human and mouse SFXNs. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model [61]. The tree with the highest log likelihood (-4427.25) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. A distance scale of 0.50 indicates 0.5 substitutions per site. This analysis involved 10 amino acid sequences. There was a total of 350 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [60]. Abbreviations: Hsa: Homo sapiens, Mmu: Mus musculus

Biological and clinical significance of SFXNs

As mentioned above, SFXNs are crucial mitochondrial membrane proteins involved in metabolite transport, mitochondrial function, and cellular metabolism. They contribute to iron homeostasis, one-carbon metabolism, and central carbon metabolism, with implications for involvement in neurodegeneration, cancer, and sideroblastic anemia.

Biological functions

SFXN1

SFXN1 is essential for serine transport from cytosol into the mitochondrial matrix, which is a key step to feed mitochondrial one-carbon metabolism [13]. One-carbon metabolism is an interconnected set of mitochondrial and cytosolic reactions that convey single-carbon units, carried by the tetrahydrofolate (THF) coenzyme, between pathways that build and modify biomolecules [30, 31]. In mitochondria, serine enters the mitochondrial branch of one-carbon metabolism, where it is cleaved to glycine in the reaction catalyzed by serine hydroxymethyltransferase which also generates 5,10-methylene-THF; the later is further metabolized to yield formate. Formate is exported to the cytosol where it fuels the folate cycle linked to thymidylate and purine synthesis as well as methylation reactions, which are critical for DNA, RNA, and protein regulation (Fig. 4) [13, 15, 30, 32]. Of note, oxidation of folate-bound one-carbon groups can also generate NADPH thus contributing to the energy production [33]. This dynamic flow of one-carbon units integrates amino acid catabolism with nucleotide biosynthesis, redox balance, and epigenetic control — functions that are essential for supporting cell growth and proliferation [13, 30].

Fig. 4.

Proposed roles for SFXNs. SFXNs transport serine or citrate to mitochondria. Whereas citrate feeds the TCA cycle for energy production, serine has multiple functions. Its main pathway is folate-dependent conversion to glycine catalyzed by SHMT2. This reaction feeds one-carbon groups to mitochondrial folate metabolism (OCM, one-carbon metabolism). These groups can be: (i) oxidized to CO2 to produce energy in the form of NADPH; (ii) converted to formate, which is exported from mitochondria to feed the cytosolic folate metabolism; and (iii) directed to formylation of Met-tRNA, which is a required (initiation) step in mitochondrial protein biosynthesis. Both serine and serine-derived glycine are also required for protein biosynthesis in mitochondria. Glycine is also required for the rate-limiting step in heme biosynthesis. Surplus glycine can be exported from mitochondria for utilization in cytosolic reactions. Proposed model also considers the putative function for SFXNs in exporting citrate from mitochondria

Absence of SFXN1 leads to serine deficiency, which limits cell proliferation in knockout (KO) cell models; however, this defect can be rescued with formate supplementation [14]. In addition, SFXN1 deficiency disrupts central carbon metabolism by depleting tricarboxylic acid (TCA) cycle intermediates, decreasing glutamate dehydrogenase activity, and altering NAD⁺/NADH ratios, as reported by Acoba and colleagues [14]. While radiolabel and reconstituted-proteoliposome assays confirmed serine as the preferred substrate, Kory et al. also observed lower-affinity transport of alanine, cysteine and glycine, indicating that SFXN1 is a serine-favoured but not serine-exclusive carrier [13].

Nonetheless, while originally characterized as a serine transporter, SFXN1 has since been implicated in the biogenesis, assembly, and functional regulation of mitochondrial respiratory complex III [13, 14, 34]. Loss or dysfunction of SFXN1 leads to selective destabilization of complex III, lowering its subunit abundance, disrupting dimerization, and suppressing enzymatic activity, but without substantially affecting the integrity of the other oxidative phosphorylation complexes [13, 14, 34].

SFXN1 also influences mitochondrial iron homeostasis indirectly by boosting glycine production. Glycine is the direct precursor for heme and, through its involvement in one-carbon metabolism, indirectly supports Fe–S-cluster biogenesis [13]. Mitochondria assemble Fe–S clusters through a tightly regulated, multistep process. Iron delivered by frataxin and sulfur extracted from cysteine by cysteine desulfurases (NFS1) via a persulfide intermediate for incorporation into nascent Fe-S clusters are combined on the Iron-Sulfur Cluster assembly protein (ISCU) scaffold to form an initial [2Fe–2 S] cluster [35]. Ferredoxin provides the necessary electrons, and the ISA1–ISA2–IBA57 complex can remodel two small clusters into a [4Fe–4 S] form essential for respiratory-chain, TCA-cycle, and DNA-repair enzymes [36, 37]. Computational analyses also suggest the presence of heme-binding motifs in SFXN1, although their functional significance remains to be determined [12].

SFXN2

Although SFXN2’s contribution to serine transport remains unclear, it is hypothesized to contribute to mitochondrial iron homeostasis [12, 13, 38]. Studies have shown that SFXN2 KO cells exhibit increased mitochondrial iron content, suggesting that SFXN2 may be involved in iron export from mitochondria, or in regulating iron utilization within the organelle [38]. However, to date, there is no evidence showing SFXN2 (or any other SFXN) to function directly as an Fe²⁺ or Fe³⁺ transporter. Increases in mitochondrial iron in SFXN2 KO cells do not result in proportional increases in heme synthesis. Instead — heme levels are decreased in these cells, and this lowering correlates with decreased activity of heme-dependent enzymes (which rely on heme as a cofactor) [38]. Interestingly, ISC-dependent enzyme activities appear unaffected in these knockout models, suggesting a selective impact on heme biosynthesis rather than broad disruptions in ISC function. Consistent with involvement in iron handling, Chen et al. [39] demonstrated that elevated SFXN2 expression in multiple-myeloma cells limited mitochondrial autophagy, enhanced iron-driven oxidative phosphorylation, and thereby promoted tumor proliferation, further underscoring the importance of SFXN2 in mitochondrial iron utilization and energy metabolism. The mechanisms underlying these changes remain unexplored, highlighting the need for further research on SFXN2’s contribution to iron and heme homeostasis [38, 39].

SFXN3

SFXN3 is thought to be involved in serine transport (similar to that of SFXN1) [13], although its exact molecular function is not fully understood. Absence of SFXN3 in SFXN3-KO mice affects the levels of certain ISC proteins, such as the Rieske protein, leading to increased mitochondrial iron content [40]. This finding suggests that SFXN3 impacts mitochondrial iron homeostasis and ISC biogenesis. Recent work by Mi et al. [41] demonstrated that SFXN3 can form a complex with poly(rC)-binding protein 2 (PCBP2) via TOM20, thereby providing a pathway for iron entry into mitochondria and reinforcing its involvement in mitochondrial iron metabolism. However, the exact mechanisms underlying SFXN3’s regulatory function and its overall impact on mitochondrial iron metabolism remain to be fully elucidated [40, 41].

SFXN4

SFXN4 is important for mitochondrial iron utilization by affecting both heme and ISC biosynthesis. These processes are essential for mitochondrial oxidative metabolism and DNA synthesis [16, 18, 42]. SFXN4 regulates heme biosynthesis by modulating ferrochelatase levels and inhibiting the translation of erythroid δ-aminolevulinic acid synthase (ALAS2), a key enzyme in the heme synthesis pathway — encoded by the ALAS2 gene and expressed exclusively in erythroid cells [16]. In addition, SFXN4 influences ISC biosynthesis by regulating the cytosolic aconitase-iron-responsive element-binding protein 1 (IRP1) switch, thereby facilitating the redistribution of iron from the cytosol to the mitochondrial matrix [10]. It is noteworthy that a recent study identified SFXN4 as a complex I assembly factor, interacting with components of the mitochondrial complex I intermediate assembly (MCIA) complex [17]. This interaction suggests that SFXN4 might participate in stabilizing and coordinating complex I integration within the mitochondrial respiratory chain [16, 18, 34].

SFXN5

SFXN5 is primarily expressed in the brain, with high levels detected across various regions, including the cerebellum, cerebral cortex, and hippocampus [8]. Whereas its precise function remains under investigation, SFXN5 has been proposed to modulate cytosolic citrate, possibly by exporting citrate from the mitochondrion [29, 43]. In most mammalian cells citrate is produced in the tricarboxylic acid cycle and exported to the cytosol by SLC25A1/CIC, providing acetyl-CoA for lipid synthesis [44]. Of note, while the export from mitochondria is viewed as the main direction of citrate transport, the entry of citrate to mitochondria through the reverse action of SLC25A1/CIC has been shown [44]. Apparently, this mechanism could direct the surplus of cytosolic citrate back to mitochondria for the energy production through the TCA cycle. Currently, however, there is no strong evidence for a dedicated mitochondrial citrate importer, but the observed reduction in cytosolic citrate in SFXN5-deficient neutrophils suggests a function for SFXN5 in modulating citrate homeostasis, potentially through export, recycling, or exchange mechanisms [29]. This hypothesis remains speculative, as the substrate specificity, transport dynamics and the directionality of SFXN5 are not yet defined. Importantly, decreased cytosolic citrate is believed to impair actin polymerization and PI [4, 5]P₂ synthesis, processes essential for proper neutrophil function [29]. The clarification of whether SFXN5 operates as a unidirectional transporter or as part of a bidirectional exchange system, as well as the participation of the transporter in balancing cytosolic and mitochondrial citrate, will require further investigation.

Tissue distribution of SFXNs

The expression of SFXNs varies across different tissues, reflecting their distinct roles in mitochondrial metabolism. According to the Human Protein Atlas (HPA), SFXN1 is expressed in most tissues but is enriched in liver, kidney, bone marrow and small intestine, all of which have high one-carbon–metabolism demand [13, 45]. SFXN2, while ubiquitously expressed, shows higher levels in bone-marrow-derived and other hematopoietic tissues, consistent with its involvement in iron homeostasis and heme biosynthesis [39, 45]. SFXN3 is predominantly expressed in neural tissues, with peak levels in cortex, hippocampus, and retina, supporting its proposed function in iron regulation in the central nervous system [40, 45]. SFXN4 exhibits high expression in tissues with high oxidative metabolism (including liver, kidney, heart and bone marrow); this corresponds to SFXN4’s function in ISC assembly and heme biosynthesis [16, 45]. Finally, SFXN5 is primarily expressed in brain, with highest expression in cerebral cortex and cerebellum, reinforcing its proposed involvement in neuronal citrate/iron metabolism [8, 45].

Clinical importance

SFXN1

SFXN1 appears to be involved in both neurodegenerative diseases, anemia, and cancer. Impaired iron regulation due to SFXN1 dysfunction is thought to contribute to anemia, because diminished iron availability disrupts hemoglobin synthesis, thereby decreasing red blood cell production and function [7, 18]. SFXN1 has been identified as a potential therapeutic target in cancer therapy [12]. Increased SFXN1 expression has been linked to breast cancer, gliomas, and lung adenocarcinoma, suggesting its involvement in tumor progression [19, 46–48]. Moreover, it has been proposed that inhibition of SFXN1 activity may slow tumor growth by disrupting serine transport and nucleotide biosynthesis [49, 50].

SFXN2

SFXN2 gene mutations leading to lowered expression of the SFXN2 transporter have been associated with multiple myeloma, whereas its overexpression has been correlated with poor patient outcomes [39]. Suppression of SFXN2 expression impedes tumor progression in myeloma xenograft models [39]; the mechanism underlying this effect likely involves alterations in mitochondrial functions and metabolic pathways essential for myeloma cell survival and proliferation. Whatever the case, SFXN2 may represent an attractive new target for the treatment of multiple myeloma. SFXN2 has also been suggested as a potential target for mitochondrial disorders (e.g., sideroblastic anemia), further emphasizing its role in metabolic regulation [12].

SFXN3

SFXN3 has been implicated in neurodegenerative diseases, particularly Parkinson and Alzheimer diseases [8, 40, 51, 52]. SFXN3 impacts synaptogenesis and synapse maintenance and is involved in α-synuclein-dependent pathways — a key factor in Parkinson disease pathology [53]. Disruptions in SFXN3 function have been linked to mitochondrial dysfunction, further associating it with neurodegenerative disease progression [54]. Similar to SFXN1 and SFXN3 gene variants that lower its iron transport capacity have also been associated with Parkinson disease [53, 54]. SFXN3 has also been identified as a potential tumor marker for oral squamous cell carcinoma (SCC) in that elevated serum anti-SFXN3 autoantibodies have been found in SCC patients; these antibodies show high sensitivity and specificity for early detection of this type of cancer [55].

SFXN4

SFXN4 gene mutations are associated with severe mitochondrial disorders, including oxidative phosphorylation deficiency syndrome (COXPD18) — which manifests with symptoms that may include macrocytic anemia, lactic acidosis, intellectual disabilities, and visual impairment [18, 42]. In addition, homozygous inactivating mutations in SFXN4 (chromosome 10q24.2) have been linked to severe macrocytic anemia with mitochondrial dysfunction and iron overload [18, 42]. Furthermore, systemic metabolic imbalances due to SFXN4 dysregulation are known to exacerbate the severity of anemia [12]. Beyond these hematologic conditions, abnormal SFXN4 activity has also been implicated in cancer patients. Mutations in the SFXN4 gene have been correlated with familial colorectal cancer [56]. SFXN4 upregulation has been observed in hepatocellular carcinoma, where it is associated with poor prognosis, thus making it a potential prognostic biomarker [57].

SFXN5

Although SFXN5’s exact function remains unclear, the SFXN5 gene — which exhibits a brain-specific expression pattern [8] — resides within the PARK3 locus on human chromosome 2p13; the physical distance between them is approximately 2.5 million base-pairs (Mb). The “PARK3 region” is a locus associated with Parkinson disease [8]. However, no studies have demonstrated any direct association of the SFXN5 gene with Parkinson disease. On the other hand, increased SFXN5 expression has been associated with breast cancer, gliomas, and lung adenocarcinoma, suggesting its involvement in these malignant disease [19, 46–48].

Structural features

SFXNs are integral membrane proteins predominantly localized in the mitochondrial inner membrane and are characterized by several structural features essential for their function. In silico analyses predict SFXNs to possess four to six transmembrane alpha helices, but there is no exact information about their orientation (Fig. 5) [12, 58]. These alpha helices form a framework that facilitates the transport of small molecules (e.g., amino acids and metabolites) across the mitochondrial membrane by most SFXNs [7, 8, 24, 58]. Nonetheless, SFXN4 has been identified as a complex I assembly factor, interacting with components of the mitochondrial complex I intermediate assembly (MCIA) complex; therefore, the structural features of SFXN4 may facilitate this assembly process, ensuring the proper formation and integration of complex I within mitochondria [17].

Fig. 5.

Mitochondrial localization of the predicted 3-dimensional structures of sideroflexins. SFXN1, SFXN3 and SFXN4 are embedded in the inner mitochondrial membrane (IMM). SFXN2 is found in either the IMM or the outer mitochondrial membrane (OMM); thus, SFXN2 is depicted in both locations. A structural model for SFXN5 is available; however, due to the lack of localization data, SFXN5 is not included in the figure. The orientation of the SFXNs in the membranes remains to be determined; therefore, they are depicted in both possible configurations. Colors on the SFXN molecules indicate the distinct secondary structures as follows: alpha-helices (magenta), beta-sheets (yellow), and loops (white). All predicted 3-dimensional structures were obtained from AlphaFold Protein Structure Database [62–64]. The figure is based on Tifoun et al. [12] and was created using Biorender (https://BioRender.com)

SFXNs possess specific conserved motifs (Fig. 2), including the highly conserved sequence of amino acids: histidine (H), proline (P), aspartate (D), and threonine (T) (known as the HPDT motif), and an asparagine-rich sequence — which ensure proper transport to the mitochondrial membrane [4, 7, 8]. The topology of the SFXN molecules involved in transport (particularly SFXN1 and SFXN3), suggests that the NH₂-terminus influences their molecular orientation and targeting within the mitochondrial inner membrane, potentially extending into the intermembrane space [14, 25]. In such a configuration, their transmembrane domains create selective pathways for substrate transport, a feature that is critical to mitochondrial function and conserved across species [7, 12, 23]. However, further experimental studies are necessary to clarify how the NH₂-terminal structure influences substrate selection across the mitochondrial membrane.

Methods

All sideroflexin gene entries were searched at NCBI Gene Entrez (accessed 10 February 2025). SFXN gene entries were selected from fourteen representative species spanning major evolutionary lineages for analysis. These included: the vertebrates Homo sapiens (human), Mus musculus (mouse), Danio rerio (zebrafish), and Xenopus tropicalis (tropical clawed frog); and the invertebrates Drosophila melanogaster (fruit fly), Centruroides sculpturatus (bark scorpion), Caenorhabditis elegans (roundworm), the cnidarian Rhopilema esculentum (jellyfish), the mollusk Magallana gigas (Pacific oyster), the echinoderm Lytechinus variegatus (green sea urchin), and the sponge Amphimedon queenslandica. Single-celled and plant representatives included Salpingoeca rosetta (choanoflagellate), Selaginella moellendorffii (lycophyte plant), and Saccharomyces cerevisiae strain S288C (yeast). All these species are summarized in Table 1. To be included, gene entries were required to meet three criteria: (a) encode a full-length protein (excluding partial or fragmented sequences), (b) have an annotated, unique chromosomal location, and (c) be classified as protein-coding, excluding known pseudogenes.

All selected SFXN nucleotide and protein entries were downloaded from NCBI Gene Entrez (accessed 10 February 2025) and cross-checked against UniProtKB/Swiss-Prot [59]. After removing partial or redundant records, the longest canonical isoform of each gene was kept for analyses. Protein sequences were aligned with Clustal Omega v1.2.4 (default settings) through the UniProt alignment interface. Conserved motifs (HPDT- and asparagine (N)-rich regions) were annotated using InterPro v96 and visualized manually. Multiple sequence alignments from the representative species panel and the human–mouse subset were used to construct maximum-likelihood phylogenetic trees with MEGA11 [60]. The Jones–Taylor–Thornton (JTT) matrix-based substitution model [61] was applied, with initial trees generated automatically using Neighbor-Join and BioNJ algorithms. Branch lengths are presented as substitutions per site. Predicted three-dimensional structures of human SFXN1–SFXN4 were retrieved from the AlphaFold Protein Structure Database [62–64]. Structures were visualized and edited in PyMOL v2.5 to highlight α-helices, β-strands, and loop regions. Schematic diagrams and figures were generated using BioRender.com.

Conclusions

The sideroflexin (SLC56) gene family represents a highly conserved group of mitochondrial transmembrane solute carriers that are involved in metabolite transport, iron homeostasis, and mitochondrial function. Over the course of evolution, these proteins have diversified to support specialized processes involved in cellular and metabolic regulation, such as serine transport (SFXN1, SFXN3), iron metabolism (SFXN2, SFXN3, SFXN4), and (likely) citrate transport (SFXN5). Their functional diversity suggests a key role in maintaining mitochondrial functional integrity and metabolic health, with direct clinical implications for involvement in neurodegenerative diseases, anemia, and cancer. However, many aspects of their transport mechanisms, regulatory pathways, and structural dynamics remain unresolved, necessitating further research. A deeper understanding of SFXN molecular functions is expected to offer novel insights into their contributions to mitochondrial disorders and disease pathogenesis.

Author contributions

VV, DWN, AIK conceived this project. AIK drafted the manuscript. DWN, DCT contributed to portions of the manuscript. AIK, DWN, DCT, VV reviewed the manuscript and provided edits. SΑK contributed to the revision of the manuscript especially in the area of one-carbon metabolism. All authors have read and approved the final manuscript.

Funding

This work was supported, in part, by the National Institutes of Health Grants AA022057 (VV) and ES033815 (VV). AIK was awarded a Fulbright scholarship to serve as a Visiting Scholar at the Department of Environmental Health Sciences, Yale School of Public Health.

Data availability

Datasets analyzed during the current study are publicly available and in the NCBI Gene Entrez database, [https://www.ncbi.nlm.nih.gov/gene].

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

VV serves as the Editor-in-Chief and DWN as a Section Editor of the Human Genomics journal. AIK, DCT, SAK declare no competing interests.