Magnesium Research 2013; 26 (4): 176-81
ORIGINAL ARTICLE
“Inside-in” or “inside-out”? The membrane topology of SLC41A1 Gerhard Sponder1,a , Katrin Rutschmann2,a , Martin Kolisek1 1 Institute of Veterinary-Physiology, Freie Universität Berlin, Berlin, Germany; 2 Dualsystems Biotech AG, Zurich-Schlieren, Switzerland; a These authors contributed equally.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
Correspondence: Martin Kolisek, Institute of Veterinary-Physiology, Free University Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
Abstract. Membrane topology is an important parameter for understanding the function and regulation of any integral protein. This aspect of the NME SLC41A1 is currently under debate. The most probable model, which has been computer-predicted, exhibits ten TMh with both termini being oriented intracellularly. However, other freely accessible online prediction programs predict that SLC41A1 possesses eleven (“outside-in” configuration), nine (“outside-in” configuration), or eight (“inside-in” configuration) TMh. The consensus based on published experimental data acquired by independent research teams is that the N-terminal flanking region is located intracellularly. However, controversy remains about the orientation of the C-terminus, which has lately been proposed to be extracellular in peer-reviewed bibliography. Here, we performed splitubiquitin functional assays with transgenic SLC41A1 fused N- or C-terminally to a Cub-LexA-VP16 reporter cassette. The bait constructs were co-expressed in S. cerevisiae st. NMY51 with positive recombinant membrane markers (Ost1, Fur4, Alg5, Tom20) tagged with NubI (or NubG). Ubiquitin could only be reconstituted if the reporter moiety was exposed to the cytosol. Functional reconstitution of ubiquitin was observed when SLC41A1 C-terminally tagged with Cub was co-expressed with NubI-tagged membrane markers, thereby, indicating a cytosolic orientation of the C-terminus of SLC41A1. Thus, our experimental data are in favor of the - the in silico analyses being strongly preferred - ten TMh model of SLC41A1 topology, with both termini being oriented intracellularly. Key words: Na+ /Mg2+ exchanger, split-ubiquitin functional assay, transmembrane helix
Abbreviations NME: Na+ /Mg2+ exchanger; SU-FA: splitubiquitin functional assay; TMh: transmembrane helix/helices; Nub: N-terminal fragment of ubiquitin; Cub: C-terminal fragment of ubiquitin
176 To cite this article: Sponder G, Rutschmann K, Kolisek M. “Inside-in” or “inside-out”? The membrane topology of SLC41A1. Magnes Res 2013; 26(4): 176-81 doi:10.1684/mrh.2014.0351
doi:10.1684/mrh.2014.0351
Na+ /Mg2+ exchanger (NME) SLC41A1 is an integral protein with a molecular mass of 56 kDa localized in the cytoplasmic membrane [16]. It is ubiquitously expressed in mammalian cells, and plays a key role in cellular Mg home-
ostasis by conducting secondary active transport (primarily Na+ -coupled Mg2+ efflux) of Mg2+ via the cytoplasmic membrane [3, 4, 7, 8]. The disturbed activity of NME has been associated with cardiovascular, metabolic, psychiatric, and neurological ailments [8, 9]. To understand the molecular basis of the possible involvement of SLC41A1 in the pathophysiology of these diseases, we need to understand its membrane topology, its structure, and the regulation of its core function. Functional studies have shown that the phosphorylation of NME mediated via cAMPactivated PKA [4, 8, 10] and potentially also PKC [3, 10] plays an important role in its regulation.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
SLC41A1 possesses ten transmembrane helices in an “inside-in” configuration
The N-terminal flanking sequence of SLC41A1 seems to be an important regulatory region [3-5] and possesses, in addition to the predicted PKC phosphorylation sites, many putative phosphorylation hotspots for p38MAPK, cdc2, GSK3, cdk5, DNAPK, and CKII (NetPhos 1.0). In our previous work, we have demonstrated experimentally that the N-terminus of SLC41A1 is oriented intracellularly [3] and, taking into consideration the most frequent computer-predicted model of SLC41A1 possessing ten TMh (in nine out of 17, freely available online prediction programs [10]), we have assumed that the C-terminus is also oriented intracellularly. The intracellular orientation of the N-terminal flanking region of SLC41A1 has subsequently been confirmed by Mandt et al. [5]. However, by employing flow cytometry analysis of C-terminally-tagged SLC41A1, they have shown that the C-terminus of SLC41A1 is oriented extracellularly, therefore, suggesting an eleven TM model of SLC41A1 topology. Here, by utilizing a split-ubiquitin functional assay [11-13], we demonstrate that SLC41A1 C-terminally tagged with the Cub-LexA-VP16 reporter moiety is targeted to the cytoplasmic membrane as such, that the C-terminus is oriented intracellularly.
Materials and methods SLC41A1 membrane topology/functional assay: SLC41A1 was cloned into the bait vectors pBT3N and pBT3-STE (LEU2 auxotrophic selection marker; both Dualsystems Biotech, Schlieren, Switzerland). Insertion led to the generation of a DNA template for the aminoterminal and carboxyterminal fusion of SLC41A1 with the Cub-LexA-VP16 reporter cassette (figures 1A and 2A). The orientation and sequence of the inserts were verified by 5’- and 3’-end sequencing. Suitable clones were transformed into Saccharomyces cerevisiae reporter strain NMY51: MATa his3delta200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ (lexAop)8-ADE2 GAL4) (Dualsystems Biotech) and co-expressed with several positive controls cloned into DUALmembrane prey vectors pAIN-x and pPR3-N (TRP1 auxotrophic selection marker; Dualsystems Biotech). Positive controls were tagged with NubI (wt) or NubG
(Ile13Gly; a non-interacting or weakly interacting Nub mutant with significantly reduced affinity for Cub) [13]. As positive controls, a selection of yeast integral membrane proteins was fused to Nub: pOst1-NubI, pOst1-NubG, (Ost1 encodes for an essential subunit of oligosaccharyltransferase complex); pFur4-NubI, pFur4-NubG, (Fur4 encodes for uracil permease); pAlg5-NubI, pAlg5-NubG (Alg5 encodes for dolichyl-phosphate beta-glucosyltransferase); pTom20-NubI, pTom20-NubG (Tom20 encodes for a central component of the mitochondrial outer membrane translocase receptor complex); and the empty vector. The strong affinity of NubI for Cub ensured that the reporter genes were activated as long as the bait was expressed and inserted into the membrane such that the CubLexA-VP16 reporter cassette was located in the cytosol.
Results and discussion Based on the 17, freely available, online predictions of SLC41A1 membrane topology, the most likely model appears to be the ten TMh model. It is predicted by nine topology prediction programs: TMPred [14], TopPred II [15], PSORT II [16], MEMSAT SVM [17-19], TOP-CONS [20], SCAMPI-seq (http://scampi.cbr. su.se/index.php?about=SCAMPI), DAS cut-off 2.2 [21], Split 4.0 [22], and TMHMM [23]. Six of these predict an “inside-in” orientation and one an “outside-out” orientation of the N- and C- termini (two do not specify the orientation). Four sets of software predict a nine TMh model and four sets of program an eleven TMh model [10]. To examine the orientation of the N- and C-termini of SLC41A1, we employed a functional assay based on the split-ubiquitin technology. Ubiquitin fused to any protein is rapidly cleaved by ubiqitin-specific proteases in vivo. Johnsson and Varshavsky have demonstrated that Cub fused to protein X can be cleaved off only if Nub is also expressed in the same cell [11]. We constructed N-terminal and C-terminal fusions of SLC41A1 with a Cub-LexA-VP16 reporter cassette (figures 1A and 2A). Both fusion constructs were placed under the control of the CYC1 promoter. NubI and NubG fragments were fused with confirmed yeast membrane proteins Ost1, Fur4, Alg5, and Tom20 such that NubI or
177
G. SPONDER, ET AL.
Alg5-Nubl Alg5-NubG
Alg5-Nubl
Tom20-NubG Tom20-Nubl
Ost1-NubG Fur4-Nubl Fur4-Nubg
D
SD-WLA empty
Ost1-NubG Fur4-Nubl Fur4-Nubg
Ost1-Nubl
Ost1-NubG
Ost1-Nubl
SD-WL
Fur4-Nubl
Ost1-Nubl
SD-WLH
Fur4-Nubg
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
SD-WLA
SD-WL
Tom20-NubG Tom20-Nubl
B
Alg5-NubG
Cub
SD-WLH
Tom20-NubG Tom20-Nubl
LexA
SLC41A1
Alg5-Nubl
VP16
Alg5-NubG
CYC1p
SD-WLA
SD-WLH
SD-WL empty
C
empty
A
Figure 1. A) DNA template for the aminoterminal fusion of SLC41A1 with the Cub-LexA-VP16 reporter cassette. B-C) show the weak reconstitution of ubiquitin in S. cerevisiae st. NMY51 coexpressing the LexA-VP16-Cub N-terminally fused to SLC41A1 (bait construct) and Ost1-NubI fusion protein (prey construct) manifested by growth of very few WLA and/or WLH prototrophic colonies growing on SD-WLA and SD-WLH media. As expected, co-expression of the bait construct with X-NubG (X stands for Ost1, Fur4, Alg5, Tom20) preys or empty prey vector (D) did not lead to growth of WLA and/or WLH prototrophs.
NubG were exposed toward the cytoplasm. NubI and NubG fusion constructs (pOst1-NubI, pOst1NubG, pFur4-NubI, pFur4-NubG, pAlg5-NubI, pAlg5-NubG, pTom20-NubI, pTom20-NubG) were placed under control of the CYC1 promoter. Respective bait and prey vectors were cotransformed into S. cerevisiae auxotrophic strain NMY51 unable to grow on synthetic minimal medium without tryptophan (W), leucine (L), histidine (H), and adenine (A). Whereas TRP1 and LEU2 serve as selective markers preventing dilution and loss of the bait and the prey plasmids, HIS3 and ADE2 are prototrophic markers produced upon activation of HIS3 and ADE2 by LexA-VP16. Figures 1B-C show that coexpres-
178
sion of the LexA-VP16-Cub N-terminally fused to SLC41A1 (bait) either did not result in the reconstitution of ubiquitin (Fur4-NubI, Alg5-NubI, Tom20-NubI; no WLA and/or WLH prototrophs were detected growing on SD-WLA and SD-WLH media) or led to weak reconstitution (Ost1-NubI; only few WLA and/or WLH prototrophic colonies were detected growing on SD-WLA and SD-WLH media), even though a strong ubiquitin reconstitution would have been expected because of the intracellular localization of the N-terminus. A likely explanation of this observation is that the length (approximately 96-99 amino acids) and the unstructured nature of the N-terminal flanking region of SLC41A1 (Sponder et al. unpublished
SLC41A1 possesses ten transmembrane helices in an “inside-in” configuration
D
SD-WLA empty
Alg5-Nubl Alg5-NubG Tom20-Nubl
Tom20-NubG
Tom20-Nubl Tom20-NubG
Ost1-Nubl Ost1-NubG Fur4-Nubl Fur4-Nubg
Ost1-NubG Fur4-Nubg
Fur4-Nubl
Ost1-Nubl
SD-WL
Ost1-NubG
Ost1-Nubl
SD-WLH
Fur4-Nubl Fur4-Nubg
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
SD-WLA
SD-WL
Tom20-NubG
r-nco-vp16
B
Tom20-Nubl
f-hind-vp16
SD-WLH
Alg5-NubG
LexA
Alg5-NubG
STE2
SD-WLA Alg5-Nubl
VP16
Cub
Alg5-Nubl
SLC41A1
SD-WLH
SD-WL empty
C
CYC1p
empty
A
Figure 2. A) DNA template for the carboxyterminal fusion of SLC41A1 with the Cub-LexA-VP16 reporter cassette. B-C) show reconstitution of ubiquitin in S. cerevisiae st. NMY51 co-expressing the LexA-VP16-Cub C-terminally fused to SLC41A1 (bait construct) and respective X-NubI fusion proteins (prey constructs; X stands for Ost1, Fur4, Alg5, Tom20) manifested by growth of WLA and/or WLH prototrophic colonies (only few colonies in case of Tom20) growing on SD-WLA and SD-WLH media. As expected, co-expression of bait construct with X-NubG preys or empty prey vector (D) did not lead to growth of WLA and/or WLH prototrophs. data) results in the inability of Cub to come into effective proximity to the NubI necessary for ubiquitin reconstitution. Figures 2B-C illustrate that the coexpression of the LexA-VP16-Cub C-terminally fused to SLC41A1 with the NubI-tagged membrane markers resulted in the reconstitution of ubiquitin (weak in the case of Tom20-NubI), and prototrophic yeast colonies were detected for all four markers on SD-WLA and/or SD-WLH cultivation media. In summary, our data indicate that the Cterminus of SLC41A1 fused with the LexA-VP16Cub reporter moiety is localized intracellularly
as it interacts with NubI fused to membrane markers allowing the reconstitution of ubiquitin and consequently restoration of WLA/H prototrophy. Therefore, our data are in favor of the ten TMh “inside-in” model of the membrane topology of SLC41A1. Mandt et al. [5] demonstrated with flow cytometry that a Myc tag fused to the C-terminus of SLC41A1 can be detected on the surface of living cells. However, our data clearly contradict their observation. Flow cytometry combined with immuno-labelling is a technique commonly used for tag-based characterization of protein topology. The split-ubiquitin functional assay on the
179
G. SPONDER, ET AL.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
other hand, is a label-free technique performed as an essential step of the split-ubiquitin twohybrid assay (SU-YTHA) [11, 13]. Both methods, flow cytometry and the split-ubiquitin functional assay, are routine techniques suitable for tag-based protein topology characterization. The discrepancies between our findings and the data of Mandt et al. are therefore, somewhat surprising. Both methods have their advantages and disadvantages and with the current knowledge base a conclusive answer to the orientation of the C-terminus of SLC41A1 cannot be given. Further experiments, employing complementary techniques such as mag-fura 2-assisted functional characterization of SLC41 C-terminally tagged with a LexA-VP16-Cub reporter cassette in yeast protoplast, are needed to clarify the membrane topology of this protein.
Acknowledgement Our gratitude is due to Martin Marak, (FU Berlin) for competent technical support, to Dr. Theresa Jones for linguistic corrections, and to Dr. Daniel Auerbach and Dr. Andreas Schulze (both Dualsystems Biotech) for critical comments on the manuscript.
Disclosure Financial support: This work was supported by a research grant from the German Research Foundation (DFG), KO-3586/3-1 and KO-3586/3-2 to MK. Conflict of interest: none.
References 1. Wabakken T, Rian E, Kveine M, Aasheim HC. The human solute carrier SLC41A1 belongs to a novel eukaryotic subfamily with homology to prokaryotic MgtE Mg2+ transporters. Biochem Biophys Res Comm 2003; 306: 718-24. 2. Goytain A, Quamme GA. Functional characterization of human SLC41A1, a Mg2+ transporter with similarity to prokaryotic MgtE Mg2+ transporters. Physiol Genomics 2005; 21: 337-42. 3. Kolisek M, Launay P, Beck A, Sponder G, Serafini N, Brenkus M, Froschauer EM, Martens H, Fleig A,
180
Schweigel M. SLC41A1 is a novel mammalian Mg2+ carrier. J Biol Chem 2008; 283: 16235-47. 4. Kolisek M, Nestler A, Vormann J, SchweigelRontgen M. Human gene SLC41A1 encodes for the Na+ /Mg2+ exchanger. Am J Physiol Cell Physiol 2012; 302: C318-26. 5. Mandt T, Song Y, Scharenberg AM, Sahni J. SLC41A1 Mg2+ transport is regulated via Mg2+ dependent endosomal recycling through its Nterminal cytoplasmic domain. Biochem J 2011; 439: 129-39. 6. Nestler A, Sponder S, Rutschmann K, Mastrototaro L, Weise C, Vormann J, Schweigel-Röntgen M, Kolisek M. Nature of SLC41A1 complexes: report on split-ubiquitin yeast two hybrid assay. Magn Res 2013; 26: 56-66. 7. Hurd TW, Otto EA, Mishima E, Gee HY, Inoue H, Inazu M, Yamada H, Halbritter J, Seki G, Konishi M, Zhou W, Yamane T, Murakami S, Caridi G, Ghiggeri G, Abe T, Hildebrandt F. Mutation of the Mg2+ transporter SLC41A1 results in a nephronophthisis-like phenotype. J Am Soc Nephrol 2013; 24: 967-77. 8. Kolisek M, Sponder G, Mastrototaro L, Smorodchenko A, Launay P, Vormann J, Schweigel-Röntgen M. Substitution p.A350V in Na+ /Mg2+ Exchanger SLC41A1, Potentially Associated with Parkinson’s Disease, Is a Gain-of-Function Mutation. PLoS ONE 2013; 8: e71096. 9. Nishizawa Y, Morii H, Durlach J. Edit. New Perspectives in Magnesium Research: Nutrition and Health. Springer-Verlag London Ltd. 2007; ISBN10: 1846283884. 10. Fleig A, Schweigel-Röntgen M, Kolisek M. Solute Carrier Family SLC41, what do we really know about it? WIREs Membrane Transport and Signaling 2013; 2: 227-39. 11. Johnsson N, Varshavsky A. Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A 1994; 91: 10340-4. 12. Stagljar I, Korostensky C, Johnsson N, te Hessen S. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 1997; 95: 5187-92. 13. Fetchko M, Stagljar I. Application of the splitubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions. Methods 2003; 32: 349-62. 14. Hofmann K, Stoffel W. TMbase - A database of membrane spanning proteins segments. Biol Chem Hoppe-Seyler. 1993; 374: 166. 15. von Heijne G. Membrane Protein Structure Prediction, Hydrophobicity Analysis and the Positiveinside Rule. J Mol Biol 1992; 225: 487-94.
SLC41A1 possesses ten transmembrane helices in an “inside-in” configuration
16. Mitsuteru C, Nakao CM, Nakai K. Improvement of PSORT II Protein Sorting Prediction for Mammalian Proteins. Genome Informatics 2002; 13: 441-2.
20. Bernsel A, Viklund H, Hennerdal A, et al. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Res 2009; 37: W465-8. 21. Cserzo M, Wallin E, Simon I, et al. Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the Dense Alignment Surface method. Prot Eng 1997; 10: 673-6.
18. Jones DT. Improving the accuracy of transmembrane protein topology prediction using evolutionary information. Bioinformatics 2007; 23: 538-44.
22. Juretic D, Zoranic L, Zucic D. Basic charge clusters and predictions of membrane protein topology. J Chem Inf Comput Sci 2002; 42: 620-32.
19. Jones DT, Taylor WR, Thornton JM. A Model Recognition Approach to the Prediction of All-Helical Membrane Protein Structure and Topology. Biochem 1994; 33: 3038-49.
23. Krogh A, Larsson B, von Heijne G, et al. Predicting Transmembrane Protein Topology with a Hidden Markov Model: Application to Complete Genomes. J Mol Biol 2001; 305: 567-80.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
17. Nugent T, Jones DT. Transmembrane protein topology prediction using support vector machines. BMC Bioinformatics 2009; 10: 159.
181
ORIGINAL ARTICLE
“Inside-in” or “inside-out”? The membrane topology of SLC41A1 Gerhard Sponder1,a , Katrin Rutschmann2,a , Martin Kolisek1 1 Institute of Veterinary-Physiology, Freie Universität Berlin, Berlin, Germany; 2 Dualsystems Biotech AG, Zurich-Schlieren, Switzerland; a These authors contributed equally.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
Correspondence: Martin Kolisek, Institute of Veterinary-Physiology, Free University Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
Abstract. Membrane topology is an important parameter for understanding the function and regulation of any integral protein. This aspect of the NME SLC41A1 is currently under debate. The most probable model, which has been computer-predicted, exhibits ten TMh with both termini being oriented intracellularly. However, other freely accessible online prediction programs predict that SLC41A1 possesses eleven (“outside-in” configuration), nine (“outside-in” configuration), or eight (“inside-in” configuration) TMh. The consensus based on published experimental data acquired by independent research teams is that the N-terminal flanking region is located intracellularly. However, controversy remains about the orientation of the C-terminus, which has lately been proposed to be extracellular in peer-reviewed bibliography. Here, we performed splitubiquitin functional assays with transgenic SLC41A1 fused N- or C-terminally to a Cub-LexA-VP16 reporter cassette. The bait constructs were co-expressed in S. cerevisiae st. NMY51 with positive recombinant membrane markers (Ost1, Fur4, Alg5, Tom20) tagged with NubI (or NubG). Ubiquitin could only be reconstituted if the reporter moiety was exposed to the cytosol. Functional reconstitution of ubiquitin was observed when SLC41A1 C-terminally tagged with Cub was co-expressed with NubI-tagged membrane markers, thereby, indicating a cytosolic orientation of the C-terminus of SLC41A1. Thus, our experimental data are in favor of the - the in silico analyses being strongly preferred - ten TMh model of SLC41A1 topology, with both termini being oriented intracellularly. Key words: Na+ /Mg2+ exchanger, split-ubiquitin functional assay, transmembrane helix
Abbreviations NME: Na+ /Mg2+ exchanger; SU-FA: splitubiquitin functional assay; TMh: transmembrane helix/helices; Nub: N-terminal fragment of ubiquitin; Cub: C-terminal fragment of ubiquitin
176 To cite this article: Sponder G, Rutschmann K, Kolisek M. “Inside-in” or “inside-out”? The membrane topology of SLC41A1. Magnes Res 2013; 26(4): 176-81 doi:10.1684/mrh.2014.0351
doi:10.1684/mrh.2014.0351
Na+ /Mg2+ exchanger (NME) SLC41A1 is an integral protein with a molecular mass of 56 kDa localized in the cytoplasmic membrane [16]. It is ubiquitously expressed in mammalian cells, and plays a key role in cellular Mg home-
ostasis by conducting secondary active transport (primarily Na+ -coupled Mg2+ efflux) of Mg2+ via the cytoplasmic membrane [3, 4, 7, 8]. The disturbed activity of NME has been associated with cardiovascular, metabolic, psychiatric, and neurological ailments [8, 9]. To understand the molecular basis of the possible involvement of SLC41A1 in the pathophysiology of these diseases, we need to understand its membrane topology, its structure, and the regulation of its core function. Functional studies have shown that the phosphorylation of NME mediated via cAMPactivated PKA [4, 8, 10] and potentially also PKC [3, 10] plays an important role in its regulation.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
SLC41A1 possesses ten transmembrane helices in an “inside-in” configuration
The N-terminal flanking sequence of SLC41A1 seems to be an important regulatory region [3-5] and possesses, in addition to the predicted PKC phosphorylation sites, many putative phosphorylation hotspots for p38MAPK, cdc2, GSK3, cdk5, DNAPK, and CKII (NetPhos 1.0). In our previous work, we have demonstrated experimentally that the N-terminus of SLC41A1 is oriented intracellularly [3] and, taking into consideration the most frequent computer-predicted model of SLC41A1 possessing ten TMh (in nine out of 17, freely available online prediction programs [10]), we have assumed that the C-terminus is also oriented intracellularly. The intracellular orientation of the N-terminal flanking region of SLC41A1 has subsequently been confirmed by Mandt et al. [5]. However, by employing flow cytometry analysis of C-terminally-tagged SLC41A1, they have shown that the C-terminus of SLC41A1 is oriented extracellularly, therefore, suggesting an eleven TM model of SLC41A1 topology. Here, by utilizing a split-ubiquitin functional assay [11-13], we demonstrate that SLC41A1 C-terminally tagged with the Cub-LexA-VP16 reporter moiety is targeted to the cytoplasmic membrane as such, that the C-terminus is oriented intracellularly.
Materials and methods SLC41A1 membrane topology/functional assay: SLC41A1 was cloned into the bait vectors pBT3N and pBT3-STE (LEU2 auxotrophic selection marker; both Dualsystems Biotech, Schlieren, Switzerland). Insertion led to the generation of a DNA template for the aminoterminal and carboxyterminal fusion of SLC41A1 with the Cub-LexA-VP16 reporter cassette (figures 1A and 2A). The orientation and sequence of the inserts were verified by 5’- and 3’-end sequencing. Suitable clones were transformed into Saccharomyces cerevisiae reporter strain NMY51: MATa his3delta200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ (lexAop)8-ADE2 GAL4) (Dualsystems Biotech) and co-expressed with several positive controls cloned into DUALmembrane prey vectors pAIN-x and pPR3-N (TRP1 auxotrophic selection marker; Dualsystems Biotech). Positive controls were tagged with NubI (wt) or NubG
(Ile13Gly; a non-interacting or weakly interacting Nub mutant with significantly reduced affinity for Cub) [13]. As positive controls, a selection of yeast integral membrane proteins was fused to Nub: pOst1-NubI, pOst1-NubG, (Ost1 encodes for an essential subunit of oligosaccharyltransferase complex); pFur4-NubI, pFur4-NubG, (Fur4 encodes for uracil permease); pAlg5-NubI, pAlg5-NubG (Alg5 encodes for dolichyl-phosphate beta-glucosyltransferase); pTom20-NubI, pTom20-NubG (Tom20 encodes for a central component of the mitochondrial outer membrane translocase receptor complex); and the empty vector. The strong affinity of NubI for Cub ensured that the reporter genes were activated as long as the bait was expressed and inserted into the membrane such that the CubLexA-VP16 reporter cassette was located in the cytosol.
Results and discussion Based on the 17, freely available, online predictions of SLC41A1 membrane topology, the most likely model appears to be the ten TMh model. It is predicted by nine topology prediction programs: TMPred [14], TopPred II [15], PSORT II [16], MEMSAT SVM [17-19], TOP-CONS [20], SCAMPI-seq (http://scampi.cbr. su.se/index.php?about=SCAMPI), DAS cut-off 2.2 [21], Split 4.0 [22], and TMHMM [23]. Six of these predict an “inside-in” orientation and one an “outside-out” orientation of the N- and C- termini (two do not specify the orientation). Four sets of software predict a nine TMh model and four sets of program an eleven TMh model [10]. To examine the orientation of the N- and C-termini of SLC41A1, we employed a functional assay based on the split-ubiquitin technology. Ubiquitin fused to any protein is rapidly cleaved by ubiqitin-specific proteases in vivo. Johnsson and Varshavsky have demonstrated that Cub fused to protein X can be cleaved off only if Nub is also expressed in the same cell [11]. We constructed N-terminal and C-terminal fusions of SLC41A1 with a Cub-LexA-VP16 reporter cassette (figures 1A and 2A). Both fusion constructs were placed under the control of the CYC1 promoter. NubI and NubG fragments were fused with confirmed yeast membrane proteins Ost1, Fur4, Alg5, and Tom20 such that NubI or
177
G. SPONDER, ET AL.
Alg5-Nubl Alg5-NubG
Alg5-Nubl
Tom20-NubG Tom20-Nubl
Ost1-NubG Fur4-Nubl Fur4-Nubg
D
SD-WLA empty
Ost1-NubG Fur4-Nubl Fur4-Nubg
Ost1-Nubl
Ost1-NubG
Ost1-Nubl
SD-WL
Fur4-Nubl
Ost1-Nubl
SD-WLH
Fur4-Nubg
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
SD-WLA
SD-WL
Tom20-NubG Tom20-Nubl
B
Alg5-NubG
Cub
SD-WLH
Tom20-NubG Tom20-Nubl
LexA
SLC41A1
Alg5-Nubl
VP16
Alg5-NubG
CYC1p
SD-WLA
SD-WLH
SD-WL empty
C
empty
A
Figure 1. A) DNA template for the aminoterminal fusion of SLC41A1 with the Cub-LexA-VP16 reporter cassette. B-C) show the weak reconstitution of ubiquitin in S. cerevisiae st. NMY51 coexpressing the LexA-VP16-Cub N-terminally fused to SLC41A1 (bait construct) and Ost1-NubI fusion protein (prey construct) manifested by growth of very few WLA and/or WLH prototrophic colonies growing on SD-WLA and SD-WLH media. As expected, co-expression of the bait construct with X-NubG (X stands for Ost1, Fur4, Alg5, Tom20) preys or empty prey vector (D) did not lead to growth of WLA and/or WLH prototrophs.
NubG were exposed toward the cytoplasm. NubI and NubG fusion constructs (pOst1-NubI, pOst1NubG, pFur4-NubI, pFur4-NubG, pAlg5-NubI, pAlg5-NubG, pTom20-NubI, pTom20-NubG) were placed under control of the CYC1 promoter. Respective bait and prey vectors were cotransformed into S. cerevisiae auxotrophic strain NMY51 unable to grow on synthetic minimal medium without tryptophan (W), leucine (L), histidine (H), and adenine (A). Whereas TRP1 and LEU2 serve as selective markers preventing dilution and loss of the bait and the prey plasmids, HIS3 and ADE2 are prototrophic markers produced upon activation of HIS3 and ADE2 by LexA-VP16. Figures 1B-C show that coexpres-
178
sion of the LexA-VP16-Cub N-terminally fused to SLC41A1 (bait) either did not result in the reconstitution of ubiquitin (Fur4-NubI, Alg5-NubI, Tom20-NubI; no WLA and/or WLH prototrophs were detected growing on SD-WLA and SD-WLH media) or led to weak reconstitution (Ost1-NubI; only few WLA and/or WLH prototrophic colonies were detected growing on SD-WLA and SD-WLH media), even though a strong ubiquitin reconstitution would have been expected because of the intracellular localization of the N-terminus. A likely explanation of this observation is that the length (approximately 96-99 amino acids) and the unstructured nature of the N-terminal flanking region of SLC41A1 (Sponder et al. unpublished
SLC41A1 possesses ten transmembrane helices in an “inside-in” configuration
D
SD-WLA empty
Alg5-Nubl Alg5-NubG Tom20-Nubl
Tom20-NubG
Tom20-Nubl Tom20-NubG
Ost1-Nubl Ost1-NubG Fur4-Nubl Fur4-Nubg
Ost1-NubG Fur4-Nubg
Fur4-Nubl
Ost1-Nubl
SD-WL
Ost1-NubG
Ost1-Nubl
SD-WLH
Fur4-Nubl Fur4-Nubg
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
SD-WLA
SD-WL
Tom20-NubG
r-nco-vp16
B
Tom20-Nubl
f-hind-vp16
SD-WLH
Alg5-NubG
LexA
Alg5-NubG
STE2
SD-WLA Alg5-Nubl
VP16
Cub
Alg5-Nubl
SLC41A1
SD-WLH
SD-WL empty
C
CYC1p
empty
A
Figure 2. A) DNA template for the carboxyterminal fusion of SLC41A1 with the Cub-LexA-VP16 reporter cassette. B-C) show reconstitution of ubiquitin in S. cerevisiae st. NMY51 co-expressing the LexA-VP16-Cub C-terminally fused to SLC41A1 (bait construct) and respective X-NubI fusion proteins (prey constructs; X stands for Ost1, Fur4, Alg5, Tom20) manifested by growth of WLA and/or WLH prototrophic colonies (only few colonies in case of Tom20) growing on SD-WLA and SD-WLH media. As expected, co-expression of bait construct with X-NubG preys or empty prey vector (D) did not lead to growth of WLA and/or WLH prototrophs. data) results in the inability of Cub to come into effective proximity to the NubI necessary for ubiquitin reconstitution. Figures 2B-C illustrate that the coexpression of the LexA-VP16-Cub C-terminally fused to SLC41A1 with the NubI-tagged membrane markers resulted in the reconstitution of ubiquitin (weak in the case of Tom20-NubI), and prototrophic yeast colonies were detected for all four markers on SD-WLA and/or SD-WLH cultivation media. In summary, our data indicate that the Cterminus of SLC41A1 fused with the LexA-VP16Cub reporter moiety is localized intracellularly
as it interacts with NubI fused to membrane markers allowing the reconstitution of ubiquitin and consequently restoration of WLA/H prototrophy. Therefore, our data are in favor of the ten TMh “inside-in” model of the membrane topology of SLC41A1. Mandt et al. [5] demonstrated with flow cytometry that a Myc tag fused to the C-terminus of SLC41A1 can be detected on the surface of living cells. However, our data clearly contradict their observation. Flow cytometry combined with immuno-labelling is a technique commonly used for tag-based characterization of protein topology. The split-ubiquitin functional assay on the
179
G. SPONDER, ET AL.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
other hand, is a label-free technique performed as an essential step of the split-ubiquitin twohybrid assay (SU-YTHA) [11, 13]. Both methods, flow cytometry and the split-ubiquitin functional assay, are routine techniques suitable for tag-based protein topology characterization. The discrepancies between our findings and the data of Mandt et al. are therefore, somewhat surprising. Both methods have their advantages and disadvantages and with the current knowledge base a conclusive answer to the orientation of the C-terminus of SLC41A1 cannot be given. Further experiments, employing complementary techniques such as mag-fura 2-assisted functional characterization of SLC41 C-terminally tagged with a LexA-VP16-Cub reporter cassette in yeast protoplast, are needed to clarify the membrane topology of this protein.
Acknowledgement Our gratitude is due to Martin Marak, (FU Berlin) for competent technical support, to Dr. Theresa Jones for linguistic corrections, and to Dr. Daniel Auerbach and Dr. Andreas Schulze (both Dualsystems Biotech) for critical comments on the manuscript.
Disclosure Financial support: This work was supported by a research grant from the German Research Foundation (DFG), KO-3586/3-1 and KO-3586/3-2 to MK. Conflict of interest: none.
References 1. Wabakken T, Rian E, Kveine M, Aasheim HC. The human solute carrier SLC41A1 belongs to a novel eukaryotic subfamily with homology to prokaryotic MgtE Mg2+ transporters. Biochem Biophys Res Comm 2003; 306: 718-24. 2. Goytain A, Quamme GA. Functional characterization of human SLC41A1, a Mg2+ transporter with similarity to prokaryotic MgtE Mg2+ transporters. Physiol Genomics 2005; 21: 337-42. 3. Kolisek M, Launay P, Beck A, Sponder G, Serafini N, Brenkus M, Froschauer EM, Martens H, Fleig A,
180
Schweigel M. SLC41A1 is a novel mammalian Mg2+ carrier. J Biol Chem 2008; 283: 16235-47. 4. Kolisek M, Nestler A, Vormann J, SchweigelRontgen M. Human gene SLC41A1 encodes for the Na+ /Mg2+ exchanger. Am J Physiol Cell Physiol 2012; 302: C318-26. 5. Mandt T, Song Y, Scharenberg AM, Sahni J. SLC41A1 Mg2+ transport is regulated via Mg2+ dependent endosomal recycling through its Nterminal cytoplasmic domain. Biochem J 2011; 439: 129-39. 6. Nestler A, Sponder S, Rutschmann K, Mastrototaro L, Weise C, Vormann J, Schweigel-Röntgen M, Kolisek M. Nature of SLC41A1 complexes: report on split-ubiquitin yeast two hybrid assay. Magn Res 2013; 26: 56-66. 7. Hurd TW, Otto EA, Mishima E, Gee HY, Inoue H, Inazu M, Yamada H, Halbritter J, Seki G, Konishi M, Zhou W, Yamane T, Murakami S, Caridi G, Ghiggeri G, Abe T, Hildebrandt F. Mutation of the Mg2+ transporter SLC41A1 results in a nephronophthisis-like phenotype. J Am Soc Nephrol 2013; 24: 967-77. 8. Kolisek M, Sponder G, Mastrototaro L, Smorodchenko A, Launay P, Vormann J, Schweigel-Röntgen M. Substitution p.A350V in Na+ /Mg2+ Exchanger SLC41A1, Potentially Associated with Parkinson’s Disease, Is a Gain-of-Function Mutation. PLoS ONE 2013; 8: e71096. 9. Nishizawa Y, Morii H, Durlach J. Edit. New Perspectives in Magnesium Research: Nutrition and Health. Springer-Verlag London Ltd. 2007; ISBN10: 1846283884. 10. Fleig A, Schweigel-Röntgen M, Kolisek M. Solute Carrier Family SLC41, what do we really know about it? WIREs Membrane Transport and Signaling 2013; 2: 227-39. 11. Johnsson N, Varshavsky A. Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A 1994; 91: 10340-4. 12. Stagljar I, Korostensky C, Johnsson N, te Hessen S. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 1997; 95: 5187-92. 13. Fetchko M, Stagljar I. Application of the splitubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions. Methods 2003; 32: 349-62. 14. Hofmann K, Stoffel W. TMbase - A database of membrane spanning proteins segments. Biol Chem Hoppe-Seyler. 1993; 374: 166. 15. von Heijne G. Membrane Protein Structure Prediction, Hydrophobicity Analysis and the Positiveinside Rule. J Mol Biol 1992; 225: 487-94.
SLC41A1 possesses ten transmembrane helices in an “inside-in” configuration
16. Mitsuteru C, Nakao CM, Nakai K. Improvement of PSORT II Protein Sorting Prediction for Mammalian Proteins. Genome Informatics 2002; 13: 441-2.
20. Bernsel A, Viklund H, Hennerdal A, et al. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Res 2009; 37: W465-8. 21. Cserzo M, Wallin E, Simon I, et al. Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the Dense Alignment Surface method. Prot Eng 1997; 10: 673-6.
18. Jones DT. Improving the accuracy of transmembrane protein topology prediction using evolutionary information. Bioinformatics 2007; 23: 538-44.
22. Juretic D, Zoranic L, Zucic D. Basic charge clusters and predictions of membrane protein topology. J Chem Inf Comput Sci 2002; 42: 620-32.
19. Jones DT, Taylor WR, Thornton JM. A Model Recognition Approach to the Prediction of All-Helical Membrane Protein Structure and Topology. Biochem 1994; 33: 3038-49.
23. Krogh A, Larsson B, von Heijne G, et al. Predicting Transmembrane Protein Topology with a Hidden Markov Model: Application to Complete Genomes. J Mol Biol 2001; 305: 567-80.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un utilisateur anonyme le 02/06/2017.
17. Nugent T, Jones DT. Transmembrane protein topology prediction using support vector machines. BMC Bioinformatics 2009; 10: 159.
181
