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The SLC45 gene family of putative sugar transporters
Molecular Aspects of Medicine 34 (2013) 655–660
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Review
The SLC45 gene family of putative sugar transporters q Olga Vitavska ⇑, Helmut Wieczorek University of Osnabrück, Faculty of Biology and Chemistry, Department of Animal Physiology, Barbarastrasse 11, 49076 Osnabrück, Germany
Guest Editor Matthias A. Hediger Transporters in health and disease (SLC series)
a r t i c l e
i n f o
Article history: Received 14 December 2011 Accepted 14 April 2012
Keywords: SLC45 Sugar transporter Sugar transport Glucose Sucrose
a b s t r a c t According to the classic point of view, transport of sugars across animal plasma membranes is performed by two families of transporters. Secondary active transport occurs via Na+ symporters of the SLC5 gene family, while passive transport occurs via facilitative transporters of the SLC2 family. In recent years a new family appeared in the scenery which was called the SLC45 gene family of putative sugar transporters, mainly because of obvious similarities to plant sucrose transporters. The SLC45 family consists of only four members that have been denominated A1–A4. These members apparently have counterparts in all vertebrates. Moreover, their amino acid sequences reveal close homologies also to respective invertebrate proteins such as a recently detected sucrose transporter in Drosophila, and suggest a phylogenetic relationship also to corresponding proteins from plants, fungi and bacteria. This minireview describes the molecular features of its members with a focus on their possible role as sugar transporters. Ó 2012 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
A novel SLC family of putative sugar transporters . SLC45A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SLC45A2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SLC45A3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SLC45A4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Drosophila Slc45-1 . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and perspectives . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. A novel SLC family of putative sugar transporters Transport of sugars across animal plasma membranes is accomplished by two groups of transport proteins, the SGLT family of Na+ symporters that belongs to the large SLC5 family consisting of more than 200 members (Wright et al., 1994; Wright Abbreviation: TMD, transmembrane domain(s). q
Publication in part sponsored by the Swiss National Science Foundation through the National Center of Competence in Research (NCCR) TransCure, University of Bern, Switzerland; Director Matthias A. Hediger; Web: http://www.transcure.ch. ⇑ Corresponding author. E-mail addresses: [email protected] (O. Vitavska), [email protected] (H. Wieczorek). 0098-2997/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mam.2012.05.014
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Table 1 A list of all members of the SLC45 gene family. For detailed information about the SLC gene tables, please visit: http://www.bioparadigms.org. Human gene name
Protein or gene name(s)
Predominant substrates
Transport type/coupling ions
Tissue distribution and cellular /subcellular expression
Link to disease
Human gene locus
Sequence accession ID
SLC45A1
Past-A, DNB5 MATP, AIM1, underwhite
Glucose, galactose
H+ symport
Brain, fetal kidney
Hypercapnia
NM_001080397.1
Skin, eye, melanocyte
OCA4, skin/hair/eye pigmentation, melanoma Prostate cancer, huntington disease
1p36.1p36.2 5p13.3
1q32.1
NM_033102.2
8q24.3
NM_001080431.1
SLC45A2
SLC45A3
Prostate, brain, kidney Brain, testis
plants
fungi
bacteria
animals
SLC45A4
Prostein
NM_016180.3
Fig. 1. Phylogenetic tree of SLC45 family like animal proteins and similar proteins from plants, fungi and bacteria. SLC45A1–4: Homo sapiens (NP_001073866.1, NP_057264.3, NP_149093.1, Q5BKX6.2); Slc45-1: Drosophila melanogaster (NP_648292.1); DP: Daphnia pulex (EFX86786.1); ZP: Zunongwangia profunda (YP_003585336.1); Gn: Glaciecola nitratireducens (AEP30121.1); Am: Alteromonas macleodii (ZP_04714820.1); Pi: Piriformospora indica (CCA68193.1); Ao: Aspergillus oryzae (XP_001818353.2); Ao: Ajellomyces capsulatus (EER44596.1); AtSUC2-4: Arabidopsis thaliana (NP_173685.1, NP_178389.1, AF175321.1). NCBI sequence accession numbers or UniProtKB/Swiss-Prot numbers are given in brackets. The tree was created using the ProtDist algorithm (Dereeper et al., 2008). Substitutions per position are indicated by the scale bar.
and Turk, 2004), and the GLUT or SLC2 family of facilitative transporters (Thorens, 1996; Thorens and Mueckler, 2010). Compared with these families, the novel SLC45 family (Table 1) is a small group which was named ‘‘putative’’ sugar transporter family mainly because one of its members had been shown to transport sugars (Shimokawa et al., 2002), and because all members exhibit an apparent amino acid sequence identity to plant sucrose transporters of slightly above 20%. A further reason for the term ‘‘putative’’ is that the four human members (A1–A4) show only 20–30% identity among each other. Nevertheless, based on derived amino acid sequences, all four members feature twelve transmembrane domains (TMD) with a large intracellular loop between TMD VI and VII, and have a signature sequence R-X-G-R-[K/R] between TMD II and III which is typical for plant sucrose transporters (Lemoine, 2000). For each of the human SLC45 genes a corresponding gene appears to exist in other mammalia which is relatively conserved amongst the diverse species. Thus human A1 and A3 proteins are about 90% identical to their murine or bovine analogs while A2 and A4 show slightly above 80% identity among these species. Recently we detected an H+/sucrose symporter in Drosophila which we called SCRT, sucrose transporter (Meyer et al., 2011). This transporter shows a significant similarity to members of the human SLC45 family (Fig. 1), and therefore it was termed by FlyBase Slc45-1. Fig. 1 indicates the apparent phylogenetic relationship of the animal proteins not only with the plant counterparts, but also with those found in bacteria and fungi. In this minireview we will give a short survey on what is known about the members of the SLC45 family and the newly discovered insect member Slc45-1.
2. SLC45A1 The human SLC45A1 gene was first published by Amler and colleagues as DNB5 (deleted in neuroblastoma-5) due to screening for putative tumor suppressors at chromosome 1p (Amler et al., 2000). In Northern blots they detected a strong signal at about 2.4 kb in fetal brain and kidney. In adult tissues a marked expression was found in brain, but transcripts were also detected in heart, muscle and kidney. Shortly afterwards the rat ortholog was detected, characterized and published as Past-A, proton-associated sugar transporter-A (Shimokawa et al., 2002). The mRNA which encodes a 751 amino acids protein
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was found to be restricted to the brain, and was not found in other adult tissues such as heart, liver, kidney, stomach, intestine or skeletal muscle. In 2005 the human gene was reassigned as SLC45A1, and the rodent orthologs as Slc45a1. Immunocytochemical analysis showed that the protein was exclusively located in the ventral surface of the medulla oblongata. Moreover the expression of the protein was strongly inducible by hypercapnia and decreased with reoxygenation, suggesting an adaptive role of Slc45a1 during a change in support of neuronal tissues with oxygen. Heterologous expression of PAST-A in COS-7 cells led to the conclusion that the protein is a plasma membrane transporter for glucose and galactose but not for fructose or sucrose. The uptake of glucose was pH dependent and increased significantly at low pH values, and it was inhibited in the presence of the protonophore CCCP (carbonyl cyanide m-chlorophenyl-hydrazone). Based on these results it is tempting to assume that Slc45a1 is an H+/sugar symporter which regulates glucose uptake into neurons of the medulla ventral surface under hypercapnia induced acid stress. 3. SLC45A2 SLC45A2 was originally identified as an antigen in human melanoma and thus published as AIM-1 (Harada et al., 2001). Shortly afterwards it was given the name MATP (membrane-associated transporter protein) for reasons of clarity because a Ser/Thr kinase had already been published using the name ‘‘AIM-1’’ and, in addition, a gene with the name ‘‘AIM1’’ (absent in melanoma 1) already existed (Newton et al., 2001). The human gene was assigned as SLC45A2 in 2005, is located on chromosome 5p and spans nearly 40 kb encoding a putative 530 amino acids protein (Newton et al., 2001). Mutations in this gene cause oculocutaneous albinism type 4 (OCA4), an autosomal recessive disorder of melanin biosynthesis that results in hypopigmentation of ocular and cutaneous tissues and can be associated with common developmental abnormalities of the eye (e.g. low visual acuity, nystagmus, strabismus, and iris transillumination). OCA4 seems to be a rare form of OCA worldwide except in Japan, where OCA4 has been found in 27% of OCA patients and as such is the second most prevalent type of OCA (Suzuki and Tomita, 2008). So far, more than 30 mutations in human SLC45A2 have been reported. Several pathogenic point mutations were found in OCA4 patients, leading to amino acid exchanges such as H38R, M42I, G44R, G64S, H94D, G100S, R101C, S143R, G198D, T302S, M335R, R348C, A501D or Y278X (Hutton and Spritz, 2008; Konno et al., 2009; Sengupta et al., 2007). Another pathogenic mutation skipped exon 2 (Newton et al., 2001). Among non-pathogenic changes in the Slc45a2 gene the single nucleotide polymorphisms E272K and F374L are the most reported ones and are, in Caucasian populations, both strongly associated with dark eyes, hair and skin, respectively (Graf et al., 2005; Nakayama et al., 2002; Newton et al., 2001; Yuasa et al. 2004; Yuasa et al., 2006). Because of its involvement in dark coloration of the skin the L374 allele was also significantly associated with protection from malignant melanoma (Fernandez et al., 2008; Guedj et al., 2008). Two polymorphisms in the SLC45A2 promoter also have been found and seem to be associated with normal Caucasian variation of pigmentation (Graf et al., 2007). Like the major pigmentation genes encoding tyrosinase, tyrosinase-related protein 1 and DOPAchrome tautomerase, the SLC45A2 gene seems to be regulated through a cAMP signaling pathway resulting in the expression modulation of MITF (microphthalmia-associated transcription factor), a transcription factor which is essential for melanocyte development (Du and Fisher, 2002). In addition, SLC45A2 expression may also be regulated due to promoter polymorphisms in this gene (Graf et al., 2007). The mouse underwhite phenotype (uw), caused by mutations in Slc45a2, the mouse ortholog of human SLC45A2, provided the first clues to the possible function of the encoded protein. So for example SLC45A2 seems to be intrinsic to melanosomes, the organelles in which melanin is synthesized and stored, and evidently affects their shape and color. Melanosomes from uw mice contain less melanin, and are less mature than organelles from wild-type mice. Furthermore, melanosomes of the mutant mice are, compared with the ovoid or round counterparts of wild type mice, smaller and crenated (Lehman et al., 2000; Sweet et al., 1998). Based on this phenomenon and on the significant similarities to plant H+/sucrose-symporters, SLC45A2 was proposed to be involved in regulation of pH as well as cellular osmolarity (Newton et al., 2001). In mouse melanoma cells, alkalinization of melanosomes caused by the adenylate cyclase activator forskolin induced melanin synthesis as well as up-regulation of genes encoding tyrosinase or the tyrosinase-related protein 1, but also of Slc45a2 (Cheli et al., 2009). The SLC45A2 mutations do not only affect tyrosinase catalytic activity but also influence sorting of other proteins in stage 2 melanosomes. For instance, uw-mutant melanocytes were shown to secrete vesicles containing tyrosinase, DOPAchrome tautomerase and tyrosinase-related protein 1 into the medium (Costin et al., 2003; Graf et al., 2005). The molecular mechanisms still are unknown. Mutations in orthologs of SLC45A2 result in reduced pigmentation in other vertebrates such as fishes and birds (medaka, chicken, Japanese quail), suggesting an important role for the encoded protein in melanin synthesis. Interestingly, expression of the SLC45A2 gene seems not to be strictly restricted to melanocytes. Thus in medaka the corresponding mRNA was found also in muscle, testis and ovary. Furthermore the Slc45a2 transcript was detected by RT-PCR also in murine kidney and uterus (Fukamachi et al., 2001). 4. SLC45A3 The human SLC45A3 gene is located on chromosome 1q32.1, and encodes a 553 amino acids protein that was originally called prostein because of its tissue-specific immunohistochemical localization in the normal and cancerous prostate, but
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not in other tissues such as brain, heart, colon or liver. Its expression appears to be androgen sensitive because mRNA as well as protein levels were up-regulated in the prostate tumor cell line LNCaP by treatment with androgens. Using Northern blots as well as microarray and real-time PCR analysis, prostein mRNA was found in the healthy prostate and in prostate tumors exclusively, although a lot of different healthy tissues and their corresponding cancer cell lines were probed (Kalos et al., 2004; Xu et al., 2001). Prostein was found in the plasma membrane, and originally the protein was predicted to have 11 TMD and an N-terminally located cleavable signal peptide (Xu et al., 2001). Since detection of prostein by FACS analysis using a monoclonal antibody directed against the loop between the last but one and the last but two TMD required cell permeabilization, this loop has to be intracellular (Kalos et al., 2004). Because the rather long N-terminus of prostein would then have to be extracellular, we have the tendency to assume that such significant differences in the SLC45A3 structure compared to other members of the SLC45 family are unlikely. Moreover, modern algorithms like HMMTOP or TMpred detect 2.0 detect 12 TMD in the derived SLC45A3 amino acid sequence. Apart from its putative role in sugar transport, several recent studies support an important role for the SLC45A3 gene in prostate cancer genesis by demonstrating gene or transcript fusions with members of the erythroblast transformation-specific (ETS) family of transcription factors. Fusions of 50 parts of SLC45A3 containing androgen specific sequence elements with ETV1, ETV5, ERG and ELK4 were shown to be androgen-regulated and up-regulated in some subsets of prostate cancer (Esgueva et al., 2010; Helgeson et al., 2008; Rickman et al., 2009; Tomlins et al., 2007). Interestingly, the SLC45A3-ELK4 transcript was also found to be up-regulated in the human epithelial-like kidney adenocarcinoma cell line ACHN (Rickman et al., 2009). 5. SLC45A4 The human SLC45A4 gene (named also KIAA1126, clone number in the Kazusa cDNA sequencing project), is located at chromosome 8q24.3 and appears to encode a protein of 768 amino acids. SLC45A4 mRNA was found in moderate amounts in human brain and testis, but was absent in lung, liver, muscle, pancreas and spleen (Hirosawa et al., 1999). Analysis using the TMHMM 2.0 algorithm predicted SLC45A4 to have a typical structure with twelve transmembrane domains, a large cytosolic loop between transmembrane domains VI and VII, and the R-X-G-R-[K/R] signature between the second and third transmembrane domain. Unfortunately any other sound information about SLC45A4 is still not available. 6. Drosophila Slc45-1 In our group we recently started to investigate Slc45-1 (CG4484) from Drosophila melanogaster, which we originally named SCRT, abbreviated from sucrose transporter (Meyer et al., 2011). As shown in Fig. 2, the 599 amino acids protein has twelve TMD as predicted by the TMHMM 2.0 algorithm, a signature sequence R-W-G-R-R between TMD II and III which is typical for sucrose transporters (Lemoine, 2000), a large intracellular loop between TMD VI and VII, and several phosphorylation sites for serine/threonine kinases. Thus Slc45-1 exhibits a significant similarity to plant sucrose transporters as well as to the members of the human SLC45 family, and with nearly 30% amino acid sequence identity it seems to be orthologous to SLC45A2.
outside
COOH
* *
* * *
NH2
inside Fig. 2. Hypothetical topology membrane spanning model of the Drosophila Slc45-1 sucrose transporter. Each circle represents one amino acid. Hydropathy analysis (TMHMM 2.0 algorithm) predicts the existence of twelve TMD with a large intracellular loop between TMD VI and VII. Gray circles with asterisks: R-W-G-R-R, corresponding to the signature sequence for sucrose transporters (Lemoine, 2000). Black circles: serines or threonines which may be targets for phosphorylation by the protein kinases A, B, C or G (GPS 2.1 Phosphorylation Predictor, high threshold). Putative phosphorylation sites in the loop between domains VI and VII are also found for all human SLC45A members, and at the N-terminus for the SLC45A members 1, 2 and 4.
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Fig. 3. Proposed function of sucrose as compatible osmolyte during melanin synthesis. After conversion of tyrosine to indole-5,6-quinone, melanin is produced by polymerization. This process decreases the solute concentration and thus the melanosome becomes hypotonic. The import of sucrose compensates for the loss of solutes and thus prevents shrinkage of the melanosome.
Experiments with yeast as a heterologous expression system revealed that Slc45-1 is a plasma membrane sucrose transporter. Since transport was pH dependent and increased with decreasing pH values and since the protonophore CCCP dissipated sucrose transport, we concluded that Slc45-1 most probably is an H+/sucrose symporter. A prominent location of Slc45-1 appeared to be, due to its close association with melanin containing structures of Drosophila embryos, the melanosomal membrane. Therefore it is tempting to speculate that Slc45-1 plays an important role during melanin biosynthesis which may be comparable to that of the human SLC45A2. In both cases sucrose could serve as a compatible osmolyte during the polymerization of tyrosine that leads to the synthesis of melanin, a process which would result, without osmotic compensation, to hypotonic melanocytes (Fig. 3). Furthermore, the fact that Slc45-1 is expressed in all development stages and that it is, as demonstrated by RNAi induced knockdown, of vital importance for the flies, suggests the involvement of SLC45-1 also in other cellular processes beyond melanin production. For instance, the finding that the protein also occurs in apical membranes of the embryonic hindgut may argue for function of Slc45-1 in sucrose uptake in the intestinal tract. 7. Conclusion and perspectives Although so far only Slc45-a1 has been demonstrated to translocate sugars across membranes, we see some evidence that also the other members of the human SLC45 gene family encode sugar transporters. The similarity with the Drosophila sucrose transporter Slc45-1 leads us to assume that at least the mammalian SLC45A2 exhibits sucrose transporting activity as we discussed above. At present it is, however, a matter of speculation whether all SLC45 members are able to transport sugars, too. Our finding that in Drosophila Slc45-1 is located also in apical gut membranes and thus could serve nutritional purposes is astonishing because sugar transport across animal membranes is widely believed to be restricted to monosaccharides. A second exciting finding is that Slc45-1 is most probably an H+ symporter. Both aspects, the transport of the disaccharide sucrose and its coupling to protons may remind by analogy of the former tenet that resorption after protein digestion only occurs via Na+ coupled transport of amino acids which was falsified not before the mid-90s when H+ coupled peptide transporters, now constituting the SLC15 family, were identified (Daniel et al., 2006). Whether or not resorption after carbohydrate digestion also could function via H+ coupled transport remains an open question. References Amler, L.C., Bauer, A., Corvi, R., Dihlmann, S., Praml, C., Cavenee, W.K., Schwab, M., Hampton, G.M., 2000. Identification and characterization of novel genes located at the t(1;15)(p36.2;q24) translocation breakpoint in the neuroblastoma cell line NGP. Genomics 64, 195–202. Cheli, Y., Luciani, F., Khaled, M., Beuret, L., Bille, K., Gounon, P., Ortonne, J.P., Bertolotto, C., Ballotti, R., 2009. {alpha}MSH and cyclic AMP elevating agents control melanosome pH through a protein kinase A-independent mechanism. J. Biol. Chem. 284, 18699–18706. Costin, G.E., Valencia, J.C., Vieira, W.D., Lamoreux, M.L., Hearing, V.J., 2003. Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4. J. Cell Sci. 116, 3203–3212. Daniel, H., Spanier, B., Kottra, G., Weitz, D., 2006. From bacteria to man: archaic proton-dependent peptide transporters at work. Physiology (Bethesda) 21, 93–102. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F., Guindon, S., Lefort, V., Lescot, M., Claverie, J.M., Gascuel, O., 2008. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469.
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The underwhite (uw) locus acts autonomously and reduces the production of melanin. J. Invest. Dermatol. 115, 601–606. Lemoine, R., 2000. Sucrose transporters in plants: update on function and structure. Biochim. Biophys. Acta 1465, 246–262. Meyer, H., Vitavska, O., Wieczorek, H., 2011. Identification of an animal sucrose transporter. J. Cell Sci. 124, 1984–1991. Nakayama, K., Fukamachi, S., Kimura, H., Koda, Y., Soemantri, A., Ishida, T., 2002. Distinctive distribution of AIM1 polymorphism among major human populations with different skin color. J. Hum. Genet. 47, 92–94. Newton, J.M., Cohen-Barak, O., Hagiwara, N., Gardner, J.M., Davisson, M.T., King, R.A., Brilliant, M.H., 2001. Mutations in the human orthologue of the mouse underwhite gene (uw) underlie a new form of oculocutaneous albinism, OCA4. Am. J. Hum. Genet. 69, 981–988. Rickman, D.S., Pflueger, D., Moss, B., VanDoren, V.E., Chen, C.X., de la Taille, A., Kuefer, R., Tewari, A.K., Setlur, S.R., Demichelis, F., Rubin, M.A., 2009. SLC45A3ELK4 is a novel and frequent erythroblast transformation-specific fusion transcript in prostate cancer. Cancer Res. 69, 2734–2738. Sengupta, M., Chaki, M., Arti, N., Ray, K., 2007. SLC45A2 variations in Indian oculocutaneous albinism patients. Mol. Vis. 13, 1406–1411. Shimokawa, N., Okada, J., Haglund, K., Dikic, I., Koibuchi, N., Miura, M., 2002. Past-A, a novel proton-associated sugar transporter, regulates glucose homeostasis in the brain. J. Neurosci. 22, 9160–9165. Suzuki, T., Tomita, Y., 2008. Recent advances in genetic analyses of oculocutaneous albinism types 2 and 4. J. Dermatol. Sci. 51, 1–9. Sweet, H.O., Brilliant, M.H., Cook, S.A., Johnson, K.R., Davisson, M.T., 1998. A new allelic series for the underwhite gene on mouse chromosome 15. J. Hered. 89, 546–551. Thorens, B., 1996. Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes. Am. J. Physiol. 270, G541–553. Thorens, B., Mueckler, M., 2010. Glucose transporters in the 21st Century. Am. J. Physiol. 298, E141–145. Tomlins, S.A., Laxman, B., Dhanasekaran, S.M., Helgeson, B.E., Cao, X.H., Morris, D.S., Menon, A., Jing, X.J., Cao, Q., Han, B., Yu, J.D., Wang, L., Montie, J.E., Rubin, M.A., Pienta, K.J., Roulston, D., Shah, R.B., Varambally, S., Mehra, R., Chinnaiyan, A.M., 2007. Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer. Nature 448, 595–599. Wright, E.M., Loo, D.D., Panayotova-Heiermann, M., Lostao, M.P., Hirayama, B.H., Mackenzie, B., Boorer, K., Zampighi, G., 1994. ‘Active’ sugar transport in eukaryotes. J. Exp. Biol. 196, 197–212. Wright, E.M., Turk, E., 2004. The sodium/glucose cotransport family SLC5. Pflügers Arch. 447, 510–518. Xu, J., Kalos, M., Stolk, J.A., Zasloff, E.J., Zhang, X., Houghton, R.L., Filho, A.M., Nolasco, M., Badaro, R., Reed, S.G., 2001. Identification and characterization of prostein, a novel prostate-specific protein. Cancer Res. 61, 1563–1568. Yuasa, I., Umetsu, K., Harihara, S., Kido, A., Miyoshi, A., Saitou, N., Dashnyam, B., Jin, F., Lucotte, G., Chattopadhyay, P.K., Henke, L., Henke, J., 2006. Distribution of the F374 allele of the SLC45A2 (MATP) gene and founder-haplotype analysis. Ann. Hum. Genet. 70, 802–811. Yuasa, I., Umetsu, K., Watanabe, G., Nakamura, H., Endoh, M., Irizawa, Y., 2004. MATP polymorphisms in Germans and Japanese: the L374F mutation as a population marker for caucasoids. Int. J. Legal Med. 118, 364–366. Olga Vitavska joined Prof. H. Wieczorek’s group in 2001 and received her Ph.D. from the University of Osnabrück in Germany in 2005, where she studied the interaction of vacuolar ATPases with actin. Afterwards she worked on the regulation of vacuolar ATPases, and recently she started research on sugar transporters. Helmut Wieczorek received his Ph.D. from Regensburg University in Germany in 1975. In 1986 he became Professor at the University of Munich, and since 1997 he is Full Professor of Animal Physiology in Osnabrück. His main research focus is on the structure, function and regulation of vacuolar ATPases, and some years ago he started to work on sugar transporters too.
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Molecular Aspects of Medicine journal homepage: www.elsevier.com/locate/mam
Review
The SLC45 gene family of putative sugar transporters q Olga Vitavska ⇑, Helmut Wieczorek University of Osnabrück, Faculty of Biology and Chemistry, Department of Animal Physiology, Barbarastrasse 11, 49076 Osnabrück, Germany
Guest Editor Matthias A. Hediger Transporters in health and disease (SLC series)
a r t i c l e
i n f o
Article history: Received 14 December 2011 Accepted 14 April 2012
Keywords: SLC45 Sugar transporter Sugar transport Glucose Sucrose
a b s t r a c t According to the classic point of view, transport of sugars across animal plasma membranes is performed by two families of transporters. Secondary active transport occurs via Na+ symporters of the SLC5 gene family, while passive transport occurs via facilitative transporters of the SLC2 family. In recent years a new family appeared in the scenery which was called the SLC45 gene family of putative sugar transporters, mainly because of obvious similarities to plant sucrose transporters. The SLC45 family consists of only four members that have been denominated A1–A4. These members apparently have counterparts in all vertebrates. Moreover, their amino acid sequences reveal close homologies also to respective invertebrate proteins such as a recently detected sucrose transporter in Drosophila, and suggest a phylogenetic relationship also to corresponding proteins from plants, fungi and bacteria. This minireview describes the molecular features of its members with a focus on their possible role as sugar transporters. Ó 2012 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
A novel SLC family of putative sugar transporters . SLC45A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SLC45A2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SLC45A3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SLC45A4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Drosophila Slc45-1 . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and perspectives . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. A novel SLC family of putative sugar transporters Transport of sugars across animal plasma membranes is accomplished by two groups of transport proteins, the SGLT family of Na+ symporters that belongs to the large SLC5 family consisting of more than 200 members (Wright et al., 1994; Wright Abbreviation: TMD, transmembrane domain(s). q
Publication in part sponsored by the Swiss National Science Foundation through the National Center of Competence in Research (NCCR) TransCure, University of Bern, Switzerland; Director Matthias A. Hediger; Web: http://www.transcure.ch. ⇑ Corresponding author. E-mail addresses: [email protected] (O. Vitavska), [email protected] (H. Wieczorek). 0098-2997/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mam.2012.05.014
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Table 1 A list of all members of the SLC45 gene family. For detailed information about the SLC gene tables, please visit: http://www.bioparadigms.org. Human gene name
Protein or gene name(s)
Predominant substrates
Transport type/coupling ions
Tissue distribution and cellular /subcellular expression
Link to disease
Human gene locus
Sequence accession ID
SLC45A1
Past-A, DNB5 MATP, AIM1, underwhite
Glucose, galactose
H+ symport
Brain, fetal kidney
Hypercapnia
NM_001080397.1
Skin, eye, melanocyte
OCA4, skin/hair/eye pigmentation, melanoma Prostate cancer, huntington disease
1p36.1p36.2 5p13.3
1q32.1
NM_033102.2
8q24.3
NM_001080431.1
SLC45A2
SLC45A3
Prostate, brain, kidney Brain, testis
plants
fungi
bacteria
animals
SLC45A4
Prostein
NM_016180.3
Fig. 1. Phylogenetic tree of SLC45 family like animal proteins and similar proteins from plants, fungi and bacteria. SLC45A1–4: Homo sapiens (NP_001073866.1, NP_057264.3, NP_149093.1, Q5BKX6.2); Slc45-1: Drosophila melanogaster (NP_648292.1); DP: Daphnia pulex (EFX86786.1); ZP: Zunongwangia profunda (YP_003585336.1); Gn: Glaciecola nitratireducens (AEP30121.1); Am: Alteromonas macleodii (ZP_04714820.1); Pi: Piriformospora indica (CCA68193.1); Ao: Aspergillus oryzae (XP_001818353.2); Ao: Ajellomyces capsulatus (EER44596.1); AtSUC2-4: Arabidopsis thaliana (NP_173685.1, NP_178389.1, AF175321.1). NCBI sequence accession numbers or UniProtKB/Swiss-Prot numbers are given in brackets. The tree was created using the ProtDist algorithm (Dereeper et al., 2008). Substitutions per position are indicated by the scale bar.
and Turk, 2004), and the GLUT or SLC2 family of facilitative transporters (Thorens, 1996; Thorens and Mueckler, 2010). Compared with these families, the novel SLC45 family (Table 1) is a small group which was named ‘‘putative’’ sugar transporter family mainly because one of its members had been shown to transport sugars (Shimokawa et al., 2002), and because all members exhibit an apparent amino acid sequence identity to plant sucrose transporters of slightly above 20%. A further reason for the term ‘‘putative’’ is that the four human members (A1–A4) show only 20–30% identity among each other. Nevertheless, based on derived amino acid sequences, all four members feature twelve transmembrane domains (TMD) with a large intracellular loop between TMD VI and VII, and have a signature sequence R-X-G-R-[K/R] between TMD II and III which is typical for plant sucrose transporters (Lemoine, 2000). For each of the human SLC45 genes a corresponding gene appears to exist in other mammalia which is relatively conserved amongst the diverse species. Thus human A1 and A3 proteins are about 90% identical to their murine or bovine analogs while A2 and A4 show slightly above 80% identity among these species. Recently we detected an H+/sucrose symporter in Drosophila which we called SCRT, sucrose transporter (Meyer et al., 2011). This transporter shows a significant similarity to members of the human SLC45 family (Fig. 1), and therefore it was termed by FlyBase Slc45-1. Fig. 1 indicates the apparent phylogenetic relationship of the animal proteins not only with the plant counterparts, but also with those found in bacteria and fungi. In this minireview we will give a short survey on what is known about the members of the SLC45 family and the newly discovered insect member Slc45-1.
2. SLC45A1 The human SLC45A1 gene was first published by Amler and colleagues as DNB5 (deleted in neuroblastoma-5) due to screening for putative tumor suppressors at chromosome 1p (Amler et al., 2000). In Northern blots they detected a strong signal at about 2.4 kb in fetal brain and kidney. In adult tissues a marked expression was found in brain, but transcripts were also detected in heart, muscle and kidney. Shortly afterwards the rat ortholog was detected, characterized and published as Past-A, proton-associated sugar transporter-A (Shimokawa et al., 2002). The mRNA which encodes a 751 amino acids protein
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was found to be restricted to the brain, and was not found in other adult tissues such as heart, liver, kidney, stomach, intestine or skeletal muscle. In 2005 the human gene was reassigned as SLC45A1, and the rodent orthologs as Slc45a1. Immunocytochemical analysis showed that the protein was exclusively located in the ventral surface of the medulla oblongata. Moreover the expression of the protein was strongly inducible by hypercapnia and decreased with reoxygenation, suggesting an adaptive role of Slc45a1 during a change in support of neuronal tissues with oxygen. Heterologous expression of PAST-A in COS-7 cells led to the conclusion that the protein is a plasma membrane transporter for glucose and galactose but not for fructose or sucrose. The uptake of glucose was pH dependent and increased significantly at low pH values, and it was inhibited in the presence of the protonophore CCCP (carbonyl cyanide m-chlorophenyl-hydrazone). Based on these results it is tempting to assume that Slc45a1 is an H+/sugar symporter which regulates glucose uptake into neurons of the medulla ventral surface under hypercapnia induced acid stress. 3. SLC45A2 SLC45A2 was originally identified as an antigen in human melanoma and thus published as AIM-1 (Harada et al., 2001). Shortly afterwards it was given the name MATP (membrane-associated transporter protein) for reasons of clarity because a Ser/Thr kinase had already been published using the name ‘‘AIM-1’’ and, in addition, a gene with the name ‘‘AIM1’’ (absent in melanoma 1) already existed (Newton et al., 2001). The human gene was assigned as SLC45A2 in 2005, is located on chromosome 5p and spans nearly 40 kb encoding a putative 530 amino acids protein (Newton et al., 2001). Mutations in this gene cause oculocutaneous albinism type 4 (OCA4), an autosomal recessive disorder of melanin biosynthesis that results in hypopigmentation of ocular and cutaneous tissues and can be associated with common developmental abnormalities of the eye (e.g. low visual acuity, nystagmus, strabismus, and iris transillumination). OCA4 seems to be a rare form of OCA worldwide except in Japan, where OCA4 has been found in 27% of OCA patients and as such is the second most prevalent type of OCA (Suzuki and Tomita, 2008). So far, more than 30 mutations in human SLC45A2 have been reported. Several pathogenic point mutations were found in OCA4 patients, leading to amino acid exchanges such as H38R, M42I, G44R, G64S, H94D, G100S, R101C, S143R, G198D, T302S, M335R, R348C, A501D or Y278X (Hutton and Spritz, 2008; Konno et al., 2009; Sengupta et al., 2007). Another pathogenic mutation skipped exon 2 (Newton et al., 2001). Among non-pathogenic changes in the Slc45a2 gene the single nucleotide polymorphisms E272K and F374L are the most reported ones and are, in Caucasian populations, both strongly associated with dark eyes, hair and skin, respectively (Graf et al., 2005; Nakayama et al., 2002; Newton et al., 2001; Yuasa et al. 2004; Yuasa et al., 2006). Because of its involvement in dark coloration of the skin the L374 allele was also significantly associated with protection from malignant melanoma (Fernandez et al., 2008; Guedj et al., 2008). Two polymorphisms in the SLC45A2 promoter also have been found and seem to be associated with normal Caucasian variation of pigmentation (Graf et al., 2007). Like the major pigmentation genes encoding tyrosinase, tyrosinase-related protein 1 and DOPAchrome tautomerase, the SLC45A2 gene seems to be regulated through a cAMP signaling pathway resulting in the expression modulation of MITF (microphthalmia-associated transcription factor), a transcription factor which is essential for melanocyte development (Du and Fisher, 2002). In addition, SLC45A2 expression may also be regulated due to promoter polymorphisms in this gene (Graf et al., 2007). The mouse underwhite phenotype (uw), caused by mutations in Slc45a2, the mouse ortholog of human SLC45A2, provided the first clues to the possible function of the encoded protein. So for example SLC45A2 seems to be intrinsic to melanosomes, the organelles in which melanin is synthesized and stored, and evidently affects their shape and color. Melanosomes from uw mice contain less melanin, and are less mature than organelles from wild-type mice. Furthermore, melanosomes of the mutant mice are, compared with the ovoid or round counterparts of wild type mice, smaller and crenated (Lehman et al., 2000; Sweet et al., 1998). Based on this phenomenon and on the significant similarities to plant H+/sucrose-symporters, SLC45A2 was proposed to be involved in regulation of pH as well as cellular osmolarity (Newton et al., 2001). In mouse melanoma cells, alkalinization of melanosomes caused by the adenylate cyclase activator forskolin induced melanin synthesis as well as up-regulation of genes encoding tyrosinase or the tyrosinase-related protein 1, but also of Slc45a2 (Cheli et al., 2009). The SLC45A2 mutations do not only affect tyrosinase catalytic activity but also influence sorting of other proteins in stage 2 melanosomes. For instance, uw-mutant melanocytes were shown to secrete vesicles containing tyrosinase, DOPAchrome tautomerase and tyrosinase-related protein 1 into the medium (Costin et al., 2003; Graf et al., 2005). The molecular mechanisms still are unknown. Mutations in orthologs of SLC45A2 result in reduced pigmentation in other vertebrates such as fishes and birds (medaka, chicken, Japanese quail), suggesting an important role for the encoded protein in melanin synthesis. Interestingly, expression of the SLC45A2 gene seems not to be strictly restricted to melanocytes. Thus in medaka the corresponding mRNA was found also in muscle, testis and ovary. Furthermore the Slc45a2 transcript was detected by RT-PCR also in murine kidney and uterus (Fukamachi et al., 2001). 4. SLC45A3 The human SLC45A3 gene is located on chromosome 1q32.1, and encodes a 553 amino acids protein that was originally called prostein because of its tissue-specific immunohistochemical localization in the normal and cancerous prostate, but
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not in other tissues such as brain, heart, colon or liver. Its expression appears to be androgen sensitive because mRNA as well as protein levels were up-regulated in the prostate tumor cell line LNCaP by treatment with androgens. Using Northern blots as well as microarray and real-time PCR analysis, prostein mRNA was found in the healthy prostate and in prostate tumors exclusively, although a lot of different healthy tissues and their corresponding cancer cell lines were probed (Kalos et al., 2004; Xu et al., 2001). Prostein was found in the plasma membrane, and originally the protein was predicted to have 11 TMD and an N-terminally located cleavable signal peptide (Xu et al., 2001). Since detection of prostein by FACS analysis using a monoclonal antibody directed against the loop between the last but one and the last but two TMD required cell permeabilization, this loop has to be intracellular (Kalos et al., 2004). Because the rather long N-terminus of prostein would then have to be extracellular, we have the tendency to assume that such significant differences in the SLC45A3 structure compared to other members of the SLC45 family are unlikely. Moreover, modern algorithms like HMMTOP or TMpred detect 2.0 detect 12 TMD in the derived SLC45A3 amino acid sequence. Apart from its putative role in sugar transport, several recent studies support an important role for the SLC45A3 gene in prostate cancer genesis by demonstrating gene or transcript fusions with members of the erythroblast transformation-specific (ETS) family of transcription factors. Fusions of 50 parts of SLC45A3 containing androgen specific sequence elements with ETV1, ETV5, ERG and ELK4 were shown to be androgen-regulated and up-regulated in some subsets of prostate cancer (Esgueva et al., 2010; Helgeson et al., 2008; Rickman et al., 2009; Tomlins et al., 2007). Interestingly, the SLC45A3-ELK4 transcript was also found to be up-regulated in the human epithelial-like kidney adenocarcinoma cell line ACHN (Rickman et al., 2009). 5. SLC45A4 The human SLC45A4 gene (named also KIAA1126, clone number in the Kazusa cDNA sequencing project), is located at chromosome 8q24.3 and appears to encode a protein of 768 amino acids. SLC45A4 mRNA was found in moderate amounts in human brain and testis, but was absent in lung, liver, muscle, pancreas and spleen (Hirosawa et al., 1999). Analysis using the TMHMM 2.0 algorithm predicted SLC45A4 to have a typical structure with twelve transmembrane domains, a large cytosolic loop between transmembrane domains VI and VII, and the R-X-G-R-[K/R] signature between the second and third transmembrane domain. Unfortunately any other sound information about SLC45A4 is still not available. 6. Drosophila Slc45-1 In our group we recently started to investigate Slc45-1 (CG4484) from Drosophila melanogaster, which we originally named SCRT, abbreviated from sucrose transporter (Meyer et al., 2011). As shown in Fig. 2, the 599 amino acids protein has twelve TMD as predicted by the TMHMM 2.0 algorithm, a signature sequence R-W-G-R-R between TMD II and III which is typical for sucrose transporters (Lemoine, 2000), a large intracellular loop between TMD VI and VII, and several phosphorylation sites for serine/threonine kinases. Thus Slc45-1 exhibits a significant similarity to plant sucrose transporters as well as to the members of the human SLC45 family, and with nearly 30% amino acid sequence identity it seems to be orthologous to SLC45A2.
outside
COOH
* *
* * *
NH2
inside Fig. 2. Hypothetical topology membrane spanning model of the Drosophila Slc45-1 sucrose transporter. Each circle represents one amino acid. Hydropathy analysis (TMHMM 2.0 algorithm) predicts the existence of twelve TMD with a large intracellular loop between TMD VI and VII. Gray circles with asterisks: R-W-G-R-R, corresponding to the signature sequence for sucrose transporters (Lemoine, 2000). Black circles: serines or threonines which may be targets for phosphorylation by the protein kinases A, B, C or G (GPS 2.1 Phosphorylation Predictor, high threshold). Putative phosphorylation sites in the loop between domains VI and VII are also found for all human SLC45A members, and at the N-terminus for the SLC45A members 1, 2 and 4.
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Fig. 3. Proposed function of sucrose as compatible osmolyte during melanin synthesis. After conversion of tyrosine to indole-5,6-quinone, melanin is produced by polymerization. This process decreases the solute concentration and thus the melanosome becomes hypotonic. The import of sucrose compensates for the loss of solutes and thus prevents shrinkage of the melanosome.
Experiments with yeast as a heterologous expression system revealed that Slc45-1 is a plasma membrane sucrose transporter. Since transport was pH dependent and increased with decreasing pH values and since the protonophore CCCP dissipated sucrose transport, we concluded that Slc45-1 most probably is an H+/sucrose symporter. A prominent location of Slc45-1 appeared to be, due to its close association with melanin containing structures of Drosophila embryos, the melanosomal membrane. Therefore it is tempting to speculate that Slc45-1 plays an important role during melanin biosynthesis which may be comparable to that of the human SLC45A2. In both cases sucrose could serve as a compatible osmolyte during the polymerization of tyrosine that leads to the synthesis of melanin, a process which would result, without osmotic compensation, to hypotonic melanocytes (Fig. 3). Furthermore, the fact that Slc45-1 is expressed in all development stages and that it is, as demonstrated by RNAi induced knockdown, of vital importance for the flies, suggests the involvement of SLC45-1 also in other cellular processes beyond melanin production. For instance, the finding that the protein also occurs in apical membranes of the embryonic hindgut may argue for function of Slc45-1 in sucrose uptake in the intestinal tract. 7. Conclusion and perspectives Although so far only Slc45-a1 has been demonstrated to translocate sugars across membranes, we see some evidence that also the other members of the human SLC45 gene family encode sugar transporters. The similarity with the Drosophila sucrose transporter Slc45-1 leads us to assume that at least the mammalian SLC45A2 exhibits sucrose transporting activity as we discussed above. At present it is, however, a matter of speculation whether all SLC45 members are able to transport sugars, too. Our finding that in Drosophila Slc45-1 is located also in apical gut membranes and thus could serve nutritional purposes is astonishing because sugar transport across animal membranes is widely believed to be restricted to monosaccharides. A second exciting finding is that Slc45-1 is most probably an H+ symporter. Both aspects, the transport of the disaccharide sucrose and its coupling to protons may remind by analogy of the former tenet that resorption after protein digestion only occurs via Na+ coupled transport of amino acids which was falsified not before the mid-90s when H+ coupled peptide transporters, now constituting the SLC15 family, were identified (Daniel et al., 2006). Whether or not resorption after carbohydrate digestion also could function via H+ coupled transport remains an open question. References Amler, L.C., Bauer, A., Corvi, R., Dihlmann, S., Praml, C., Cavenee, W.K., Schwab, M., Hampton, G.M., 2000. Identification and characterization of novel genes located at the t(1;15)(p36.2;q24) translocation breakpoint in the neuroblastoma cell line NGP. Genomics 64, 195–202. Cheli, Y., Luciani, F., Khaled, M., Beuret, L., Bille, K., Gounon, P., Ortonne, J.P., Bertolotto, C., Ballotti, R., 2009. {alpha}MSH and cyclic AMP elevating agents control melanosome pH through a protein kinase A-independent mechanism. J. Biol. Chem. 284, 18699–18706. Costin, G.E., Valencia, J.C., Vieira, W.D., Lamoreux, M.L., Hearing, V.J., 2003. Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4. J. Cell Sci. 116, 3203–3212. Daniel, H., Spanier, B., Kottra, G., Weitz, D., 2006. From bacteria to man: archaic proton-dependent peptide transporters at work. Physiology (Bethesda) 21, 93–102. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F., Guindon, S., Lefort, V., Lescot, M., Claverie, J.M., Gascuel, O., 2008. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469.
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Afterwards she worked on the regulation of vacuolar ATPases, and recently she started research on sugar transporters. Helmut Wieczorek received his Ph.D. from Regensburg University in Germany in 1975. In 1986 he became Professor at the University of Munich, and since 1997 he is Full Professor of Animal Physiology in Osnabrück. His main research focus is on the structure, function and regulation of vacuolar ATPases, and some years ago he started to work on sugar transporters too.