SLC24A5 Encodes a trans-Golgi Network Protein with Potassium-dependent Sodium-Calcium Exchange Activity That Regulates Human Epidermal Melanogenesis
2007; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês
10.1074/jbc.m707521200
ISSN1083-351X
AutoresRebecca S. Ginger, Sarah E. Askew, Richard M. Ogborne, Stephen W. Wilson, Dudley Ferdinando, Tony Dadd, Adrian Smith, Shubana Kazi, Robert T. Szerencsei, Robert J. Winkfein, Paul P. M. Schnetkamp, Martin R. Green,
Tópico(s)RNA regulation and disease
ResumoA non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of ∼43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity. A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of ∼43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity. For many years the genetic determinants of human skin color have attracted interest from both pigmentation biologists and anthropologists (1Sturm R.A. Teasdale R.D. Gene (Amst.). 2001; 277: 49-62Crossref PubMed Scopus (318) Google Scholar, 2Shriver M.D. Parra E.J. Dios S. Bonilla C. Norton H. Jovel C. Pfaff C. Jones C. Massac A. Cameron N. Baron A. Jackson T. Argyropoulos G. Jin L. Hoggart C.J. McKeigue P.M. Kittles R.A. Hum. Genet. 2003; 112: 387-399Crossref PubMed Scopus (422) Google Scholar, 3Barsh G.S. PLoS Biol. 2003; 1: E27Crossref PubMed Scopus (119) Google Scholar, 4Izagirre N. Garcia I. Junquera C. de la Rua C. Alonso S. Mol. Biol. Evol. 2006; 23: 1697-1706Crossref PubMed Scopus (90) Google Scholar, 5Jackson I.J. Eur. J. Hum. Genet. 2006; 14: 979-980Crossref PubMed Scopus (5) Google Scholar). Using high density whole genome array technology, we have defined the key genetic variants associated with natural skin color variation in a population of South Asian ancestry (6Stokowski R.P. Pant P.V.K. Dadd T. Fereday A. Hinds D.A. Jarman C. Filsell W. Ginger R.S. Green M.R. van der Ouderaa F.J. Cox D.R. Am. J. Hum. Genet. 2007; 81: 1119-1132Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). We identified a non-synonymous single nucleotide polymorphism (nsSNP) 2The abbreviations used are:nsSNPnon-synonymous single nucleotide polymorphismNHMnormal human epidermal melanocytesNCKXpotassium-dependent sodium calcium exchangersiRNAsmall interfering RNAbis-tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPBSphosphate-buffered salineHEK cellshuman embryonic kidney cellsTGNtrans-Golgi network. in the SLC24A5 gene (rs1426654) that was previously unknown to skin pigmentation biologists. Independently, a group of zebrafish researchers discovered that a different mutation in the zebrafish orthologue of this gene was responsible for the hypo-pigmented phenotype of the golden zebrafish (7Lamason R.L. Mohideen M.A. Mest J.R. Wong A.C. Norton H.L. Aros M.C. Jurynec M.J. Mao X. Humphreville V.R. Humbert J.E. Sinha S. Moore J.L. Jagadeeswaran P. Zhao W. Ning G. Makalowska I. McKeigue P.M. O'Donnel D. Kittles R. Parra E.J. Mangini N.J. Grunwald D.J. Shriver M.D. Canfield V.A. Cheng K.C. Science. 2005; 310: 1782-1786Crossref PubMed Scopus (822) Google Scholar). Using data from the human HapMap project (8Consortium International HapMap Nature. 2005; 437: 1299-1320Crossref PubMed Scopus (4838) Google Scholar), they observed that the alternate alleles of the nsSNP (rs1426654) in human SLC24A5 were present at very different frequencies in populations of African and European ancestry. Furthermore, they demonstrated that the allele encoding threonine 111 of the protein encoded by SLC24A5 was associated with lighter skin in an admixed African-American population. Subsequent analyses have pinpointed this SNP and another in the SLC45A2 gene as participants in the evolution of light skin in Europeans but not East Asians (9Norton H.L. Kittles R.L. Parra E. McKeigue P. Mao X. Cheng K. Canfield V.A. Bradley D.G. McEvoy B. Shriver M.D. Mol. Biol. Evol. 2006; 24: 710-722Crossref PubMed Scopus (298) Google Scholar, 10Myles S. Somel M. Tang K. Kelso J. Stoneking M. Hum. Genet. 2007; 120: 613-621Crossref PubMed Scopus (91) Google Scholar, 11Soejima M. Koda Y. Int. J. Med. 2007; 121: 36-39Google Scholar). non-synonymous single nucleotide polymorphism normal human epidermal melanocytes potassium-dependent sodium calcium exchanger small interfering RNA 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol phosphate-buffered saline human embryonic kidney cells trans-Golgi network. Amino acid sequence alignments predict that SLC24A5 encodes a member of the potassium-dependent sodium calcium exchanger protein family, designated NCKX5. The NCKX family currently consists of five members (NCKX 1-5) that are thought to use both the inward sodium gradient and the outward potassium gradient to extrude calcium across the plasma membrane. NCKX proteins have been shown to operate in retinal rod and cone photoreceptors as well as in several types of neurons in the brain (for review, see Ref. 12Altimimi H.F. Schnetkamp P.P.M. Channels. 2007; 1: 62-69Crossref PubMed Scopus (51) Google Scholar). A sixth protein, designated NCKX6, is now thought to belong to a distinct, but related protein family (CCX) as it shows substantial divergence in phylogenetic analyses (13Cai X. Lytton J. Mol. Biol. Evol. 2004; 21: 1692-1703Crossref PubMed Scopus (187) Google Scholar) and lacks many of the residues conserved between NCKX1-5 that are key for transport activity. Although SLC24A5 is predicted to encode an NCKX protein, the exchanger activity of NCKX5 has yet to be experimentally demonstrated. The residue altered by the nsSNP in SLC24A5 lies in a region of the protein that is highly conserved across all NCKX family members. This region contains a number of residues that are critical for exchange function (14Winkfein R.J. Szerencsei R.T. Kinjo T.G. Kang K. Perizzolo M. Eisner L. Schnetkamp P.P. Biochemistry. 2003; 42: 543-552Crossref PubMed Scopus (52) Google Scholar, 15Kang K.J. Kinjo T.G. Szerencsei R.T. Schnetkamp P.P. J. Biol. Chem. 2005; 280: 6823-6833Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 16Kinjo T.G. Kang K. Szerencsei R.T. Winkfein R.J. Schnetkamp P.P. Biochemistry. 2005; 44: 7787-7795Crossref PubMed Scopus (15) Google Scholar), and the experimentally determined topology for NCKX2 suggests that the polymorphic residue of NCKX5 sits in a transmembrane domain (17Kinjo T.G. Szerencsei R.T. Winkfein R.J. Kang K. Schnetkamp P.P. Biochemistry. 2003; 42: 2485-2491Crossref PubMed Scopus (43) Google Scholar). NCKX1-4 are located in the plasma membrane, yet surprisingly, overexpressed hemagglutinin-tagged recombinant zebrafish NCKX5 could not be detected in the plasma membrane of MNT1 cells (7Lamason R.L. Mohideen M.A. Mest J.R. Wong A.C. Norton H.L. Aros M.C. Jurynec M.J. Mao X. Humphreville V.R. Humbert J.E. Sinha S. Moore J.L. Jagadeeswaran P. Zhao W. Ning G. Makalowska I. McKeigue P.M. O'Donnel D. Kittles R. Parra E.J. Mangini N.J. Grunwald D.J. Shriver M.D. Canfield V.A. Cheng K.C. Science. 2005; 310: 1782-1786Crossref PubMed Scopus (822) Google Scholar), and NCKX5 has been detected in fractionated melanocytes enriched for a subcellular organelle, the melanosome, using sucrose gradient fractionation and proteomic analysis (18Chi A. Valencia J.C. Hu Z.Z. Watabe H. Yamaguchi H. Mangini N.J. Huang H. Canfield V.A. Cheng K.C. Yang F. Abe R. Yamagishi S. Shabanowitz J. Hearing V.J. Wu C. Appella E. Hunt D.F. J. Proteome Res. 2006; 5: 3135-3144Crossref PubMed Scopus (160) Google Scholar). Here we describe a full analysis of the transcript expression profile of all known sodium-calcium exchangers in cultured human and murine melanocytes together with an immunocytochemical analysis of native NCKX5 expression. We show that this protein has a novel trans-Golgi network intracellular localization in normal human epidermal melanocytes (NHM), and using siRNA-mediated knockdown we demonstrate conclusively that SLC24A5 expression is required for melanin synthesis in both cultured mouse and human melanocytes. We also demonstrate that, as predicted, SLC24A5 encodes a protein with potassium-dependent sodium-calcium exchanger activity. Through site-directed mutagenesis in a heterologous expression system, we demonstrate that the residue switch encoded by the nsSNP in SLC24A5 markedly alters exchanger activity when introduced into NCKX2 or NCKX5. Together, these data support the hypothesis that a nsSNP in the SLC24A5 gene directly alters human skin color through its effect on the sodium-calcium exchanger activity of NCKX5. Cell Culture—NHM from neonatal foreskin were cultured in MGM (M254 medium supplemented with human melanocyte growth supplement) (Cascade Biologics) at 37 °C, 9% CO2. Melanocytes were dedifferentiated as previously described (19Cook A.L. Donatien P.D. Smith A.G. Murphy M. Jones M.K. Herlyn M. Bennett D.C. Leonard J.H. Sturm R.A. J. Investig. Dermatol. 2003; 121: 1150-1159Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) using M254 and 50 nm EDN3 for 7 days before knockdown (cells denoted MC:MB). B16 F10 mouse melanoma cells (ATCC) were cultured in minimum essential medium with Earle's salts supplemented with 10% fetal calf serum and 2 mm l-glutamine at 37 °C, 5% CO2. Real-time PCR—Total RNA was extracted using the RNeasy mini kit (Qiagen) and quantified using Ribo Green (Invitrogen). 1 μg of total RNA was reverse-transcribed using the Roche Applied Science first strand synthesis kit. Real-time PCR was performed on a Bio-Rad iCycler using human or murine SLC24 or SLC8, human vSNARE, or murine TBP QuantiTect real-time PCR primers (Qiagen) and SYBR Green PCR master mix (Bio-Rad). All measurements from each cell type were made on a single cDNA sample. Data analysis was performed using the comparative cycle threshold method (ΔCT). Anti-NCKX5 Antibodies—Peptides anti-NCKX5-(I) (DEGQPFIRRQSRTDSG) and anti-NCKX5-(C) (GNNKIRGCGG) were coupled to keyhole limpet hemocyanin, and rabbits were immunized in a 90-day protocol. Bleeds were affinity-purified using individual peptides conjugated to bovine serum albumin on a Sepharose support. Immunoblotting—For membrane extracts NHM were lysed in 0.25 m sucrose, PBS using a Dounce homogenizer (30 strokes). Homogenates were centrifuged at 20,000 × g, 20 min, 4 °C. Membrane-associated proteins were extracted using 1% Triton X-100 in PBS plus protease inhibitor mixture and centrifuged. Alternatively, whole cell lysates were prepared from NHM using 1% Triton X-100 in PBS plus protease inhibitor mixture, and cell debris was removed by centrifugation. Protein content was determined by BCA assay. Proteins (15 μg/well unless otherwise stated) were separated by SDS-PAGE on 10% Novex bis-tris acrylamide gels, transferred to Invitrulon membrane, blocked in PBS, 0.1% Tween 20 with 2% w/v skimmed milk protein (PBS/T), and probed with primary antibody diluted in PBS/T. Primary antibodies consisted of anti-NCKX5-(C) at 0.5 μg/ml goat anti-Trp1 (G17), mouse anti-tyrosinase clone T311, mouse anti-Lamp-1 clone H4A3, mouse anti-Mart-1 clone A103 (all from Santa Cruz Biotechnology, Inc.), mouse HMB45 (against pmel17) clone MCA2146 (Serotec), and mouse anti-β-actin (Abcam Plc). Secondary antibody was horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG (Jackson ImmunoResearch) as appropriate diluted in PBS/T. Detection was via SuperSignal Western picochemiluminescence substrate (Perbio) and visualized with a Chemidoc XRS imaging system (Bio-Rad). The Mr of the bands was determined using Quantity One 4.6.3 one-dimensional analysis software from Bio-Rad. Myc-tagged hNCKX5 was expressed by transient transfection in HEK293 cells. Deglycosylation with peptide N-glycosidase F was carried out as described before (17Kinjo T.G. Szerencsei R.T. Winkfein R.J. Kang K. Schnetkamp P.P. Biochemistry. 2003; 42: 2485-2491Crossref PubMed Scopus (43) Google Scholar). Immunofluorescence Microscopy—Cells grown on glass coverslips were fixed with 2% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 0.5% saponin. Cells were blocked with 0.2% bovine serum albumin, 0.1% saponin, PBS for 1 h at room temperature, probed with primary and secondary antibodies, and mounted with Vectashield® (Vector Laboratories). Primary antibodies were rabbit anti-human NCKX5-(I) and NCKX5-(C) at 0.8 μg/ml, TGN46 and HMB45 (AbD Serotec), GM130 (BD Biosciences), anti-gp100 (AbCam), Mel-5 anti-TYRP1 (Signet), and T311 anti-tyrosinase (AbCam). Other antibodies tested for co-localization were Lamp 1, flotillin 2 and syntaxin 8 (BD Biosciences), EEA1, Sar1, β-COP, PEX-19 (AbCam), Lamp 2 (RDI), C423 (anti-clathrin L) (Covance), and syntaxin 6 (Sigma-Aldrich). Secondary antibodies were Alexa Fluor® 488 donkey anti-rabbit, 633 donkey anti-sheep, or 633 goat anti-mouse (Invitrogen). Confocal microscopy was performed using a Leica TCSSp1 confocal S laser microscope with Leica LCS software. Images were acquired using 20× (dry) or 60× (oil immersion 1.4na) objectives. 488- and 633-nm excitation was via argon or HeNe lasers respectively. Optical series were collected at 0.5 μm z-steps. For co-localizations with GM130, the topological separation of the signal from each channel was determined by plotting the intensity profile of each signal in three separate cross-sections of the Golgi stacks. RNA-mediated Interference Transfections—B16 or NHM were seeded at 2 × 104 cells/cm2 for melanogenesis experiments or 1 × 104 cells/cm2 for immunofluorescence microscopy experiments; MC:MB cells were seeded at 1.5 × 104 cells/cm2. Cells were adhered for 24 h and transfected with 2 μg/ml Lipofectamine 2000 and 50 nm or 100 nm Stealth™ RNA-mediated interference duplex. All dilutions were performed with Opti-MEM. Murine siRNA targeted nucleotides 762-787, with a corresponding scrambled control. Human siRNA targeted nucleotides 185-210 and 492-517 with scrambled control corresponding to 260-285. Cells were incubated with transfection reagents for 6-8 h. NHM were returned to MGM and grown for 5-6 days before analysis. B16 cells were grown in phenol red-free Dulbecco's modified Eagle's medium, 10% fetal calf serum, 4 mm l-glutamine, after 72 h the media was analyzed for melanin content, and the cells incubated for 1 h in WST-1 reagent (Roche Applied Science) to determine viability. Sucrose Density Gradient Fractionation—Light and Dark NHM were extracted to obtain a total granule fraction and fractionated essentially as described by Chen et al. (23Chen K. Manga P. Orlow S.J. Mol. Biol. Cell. 2002; 13: 1953-1964Crossref PubMed Scopus (112) Google Scholar). Briefly, cells were washed, trypsinized, and resuspended in 0.25 m homogenization buffer. After 60 strokes in a Dounce tissue-grinder, they were centrifuged at 100 × g to remove debris, and the supernatant was centrifuged at 100,000 × g (4 °C) before loading onto a stepped sucrose gradient (0.8-2 m in 0.2 m increments). Gradients were centrifuged at 25,000 rpm (4 °C) in a Beckman SW28 rotor. Interface fractions were collected, and 7.5 μl of each fraction was loaded per well in SDS reducing buffer. Melanin Assay—NHM were lysed in 1% Triton X-100, PBS and centrifuged at 15,000 g for 10 min, and protein was quantified by BCA assay. Melanin pellets washed in 1:1 ethanol/diethyl ether were dissolved in 1 m NaOH, 10% Me2SO, 30 min, 60 °C. Melanin content of the supernatants or culture media was measured at 450 nm. Clustal Alignments—Protein sequences were aligned using ClustalW program using the default settings. The resultant alignment files were visualized using Jalview multiple alignment editor (20Clamp M. Cuff J. Searle S.M. Barton G.J. Bioinformatics. 2004; 20: 426-427Crossref PubMed Scopus (1229) Google Scholar), and manual adjustments made as necessary. Functional Analysis of Mutant NCKX2 and NCKX5—Codons for the indicated residues were mutated using cDNA of the Myc-tagged short splice variant of human NCKX2 (AAF25811) or human NCKX5 and cloned in to the pEIA vector as described before (14Winkfein R.J. Szerencsei R.T. Kinjo T.G. Kang K. Perizzolo M. Eisner L. Schnetkamp P.P. Biochemistry. 2003; 42: 543-552Crossref PubMed Scopus (52) Google Scholar). The Myc tag (EQKLISEEDL) was inserted between Ser-52 and Glu-53 of human NCKX5. The mutant and wild-type NCKX2 and NCKX5 proteins were expressed in insect High Five cells, and protein expression was monitored by Western blotting with the Myc mAb (New England Biolabs). NCKX function was assayed by measuring 45Ca2+ uptake via reverse exchange in Na+-loaded cells as described in detail previously (21Szerencsei R.T. Tucker J.E. Cooper C.B. Winkfein R.J. Farrell P.J. Iatrou K. Schnetkamp P.P. J. Biol. Chem. 2000; 275: 669-676Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 14Winkfein R.J. Szerencsei R.T. Kinjo T.G. Kang K. Perizzolo M. Eisner L. Schnetkamp P.P. Biochemistry. 2003; 42: 543-552Crossref PubMed Scopus (52) Google Scholar). External 45Ca2+ was removed by a rapid filtration/washing procedure using borosilicate glass fiber filters (21Szerencsei R.T. Tucker J.E. Cooper C.B. Winkfein R.J. Farrell P.J. Iatrou K. Schnetkamp P.P. J. Biol. Chem. 2000; 275: 669-676Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). NaCl, KCl, LiCl, and choline chloride were all Sigma Ultra grade. Protein content of cell samples was determined with the Bio-Rad protein assay. SLC24A5 mRNA Is the Predominant Sodium-Calcium Exchanger Transcript in Human and Mouse Melanocytes—In mammalian cells sodium-calcium exchange is mediated both through NCKX proteins, encoded by the SLC24 gene family, and potassium-independent sodium-calcium exchanger (NCX) proteins encoded by the closely related, but distinct SLC8 gene family. This gene family comprises three members, NCX1-3, transcripts of which are found in many tissues, in particular in the heart and brain (for review, see Ref. 22Annunziato L. Pignataro G. Boscia F. Sirabella R. Formisano L. Saggese M. Cuomo O. Gala R. Secondo A. Viggiano D. Molinaro P. Valsecchi V. Tortiglione A. Adornetto A. Scorziello A. Cataldi M. Di Renzo G.F. Ann. N. Y. Acad. Sci. 2007; 1099: 413-426Crossref PubMed Scopus (39) Google Scholar). The transcript profile of all known sodium-calcium (potassium) exchangers was determined in monolayer cultures of NHM and mouse melanocytes using real-time PCR (Fig. 1). In both cultures the predominant isoform was SLC24A5, which was >100-fold more highly expressed than any of the other SLC24A transcripts, and transcripts of the SLC8 family were not detected in either cell line. In NMH low levels of SLC24A1 and very low levels of SLC24A3 and SLC24A4 were detected, and in the mouse melanocytes, low levels of SLC24A4 and very low levels of SLC24A1 and SLC24A3 were found. NCKX5 Protein Is Located in an Intracellular Membrane and Partially Co-localizes with the Trans-Golgi Network—The specificity of affinity-purified antisera for native NCKX5 protein was assessed through siRNA-mediated knockdown in NHM. Transfection with two different siRNA duplexes (siRNA 185 and siRNA 492) resulted in >90% knockdown of SLC24A5 transcript levels compared with control cultures after 48 h. 3R. S. Ginger, S. E. Askew, R. M. Ogborne, S. Wilson, D. Ferdinando, T. Dadd, A. M. Smith, S. Kazi, R. T. Szerencsei, R. J. Winkfein, P. P. M. Schnetkamp, and M. R. Green. Western blots of cell extracts harvested 5 days after transfection and probed with affinity-purified anti-NCKX5-(C) sera showed that a triplet of bands of ∼43 kDa were clearly detected in extracts from control or untreated cells but were virtually absent in extracts from SLC24A5 siRNA-treated cells (Fig. 2A). The apparent size of these bands is surprisingly low compared with the predicted molecular mass in the absence of glycosylation of 54.9 kDa. However, the lower molecular mass observed here is consistent with that observed for heterologously expressed recombinant hNCKX5 detected through amino and carboxyl-terminal tags, which also runs as a triplet of bands ∼43 kDa (Fig. 2B). De-glycosylation of the recombinant protein (Fig. 2B) removes the upper band, suggesting that the lower bands reflect signal peptide cleavage of unglycosylated protein. Non-transfected HEK cells do not show any reactivity with the anti-Myc antibody. 3R. S. Ginger, S. E. Askew, R. M. Ogborne, S. Wilson, D. Ferdinando, T. Dadd, A. M. Smith, S. Kazi, R. T. Szerencsei, R. J. Winkfein, P. P. M. Schnetkamp, and M. R. Green. These results show that the affinity-purified polyclonal antibody preparation detects NCKX5 protein in human melanocyte extracts, with some nonspecific reactivity, suggested by the detection of other higher and lower molecular weight bands that are not affected by SLC24A5 knockdown. Cultured dark NHM probed by immunofluorescence with either of our anti-NCKX5 antisera, raised against different peptides and regions of NCKX5, showed the same strong perinuclear staining and also punctuate staining distributed throughout the cytoplasm (Fig. 2C). We found no evidence of plasma membrane staining with these antisera using confocal microscopy. A similar pattern of staining was observed in both light and dark NHM, although the perinuclear staining was less intense in lighter melanocytes.3 The perinuclear staining co-localized with the trans-Golgi marker TGN46 (Fig. 2C), although merged images of serial optical sections revealed that NCKX5 only co-localized with part of the TGN (Fig. 3A). Co-staining of NHM with anti-NCKX5 and the cis-Golgi marker GM130 (Fig. 3B) showed that the location of the two antigens was close though not coincident with a separation of ∼300-500 nm, calculated from plots of signal intensity in each channel across multiple sections the Golgi stacks.3 These data suggest that NCKX5 is partially located in a region of the TGN. Treatment of light or dark melanocytes with either of the human siRNA duplexes for 5 days resulted in complete loss of perinuclear NCKX5 in >90% of cells. This is seen as a loss of yellow color in the merged images of cells double-stained for NCKX5 (green) and TGN46 (red). However no marked effect on the punctate staining was observed (Fig. 3C).3 This confirms that the perinuclear staining we have observed with both antibody preparations is specific to NCKX5 and not due to the nonspecific reactivity detected by Western blot. However the punctuate staining is either not specific to this protein, or the protein localized to these structures is not removed by siRNA knockdown even after 10 days.3 Co-localization studies with multiple markers of melanosomes, lysosomes, or endosomes were unable to determine the identity of this punctuate staining (Fig. 3D).3 NCKX5 Co-fractionates with TGN46 in NHM Extracts—Subcellular fractionation provides an alternative approach to determine the localization of proteins. Sucrose density gradient centrifugation has previously been used to enrich for melanosomal proteins (23Chen K. Manga P. Orlow S.J. Mol. Biol. Cell. 2002; 13: 1953-1964Crossref PubMed Scopus (112) Google Scholar). Using the same approach, fractionated NHM extracts were probed with anti-NCKX5 (C) and the TGN46 antibody (Fig. 4). Fractions 4-6 (corresponding to the 1.2/1.4 m, 1.4/1.6 m, and 1.6/1.8 m sucrose interfaces, respectively) contained pigment and were similarly enriched for both NCKX5 and TGN46, supporting the co-localization observed between these two antigens using immunofluorescence and indicating that these fractions contain trans-Golgi membranes as well as melanosomes. SLC24A5 Knockdown Reduces Melanin Production in Mouse B16 Melanocytes—In melanocyte monolayer cultures, where transfer is not possible, melanosome turnover is limited to some degree by cellular turnover, making it difficult to measure the inhibition of melanin production over short time-courses. Mouse B16 cells are unusual in this respect because they secrete melanin directly into the surrounding culture media, and melanogenesis is only triggered when cells reach the stationary phase of growth (24Laskin J.D. Piccinini L. Engelhardt D.L. Weinstein I.B. J. Cell. Physiol. 1982; 113: 481-486Crossref PubMed Scopus (36) Google Scholar). This affords the opportunity to assess the effect of SLC24A5 knockdown against a low background of pre-existing melanin. Therefore, the effect of SLC24A5 siRNA knockdown on melanin production was first assessed in mouse B16 melanocytes. Treatment with siRNA 762 elicited a marked and reproducible reduction in melanin production 72 h post-knockdown (87.7% reduction p < 0.0001) compared with the control siRNA (Fig. 5, A and B) without any detectable toxicity. This demonstrates that this gene and its products play a controlling role in melanogenesis in this system. SLC24A5 Knockdown Reduces Melanin Production in NHM—The effect of SLC24A5 knockdown on melanogenesis in cultured NHM was investigated by comparing the melanin content of cell pellets 5 days after siRNA transfection. NHM transfected with either of the SLC24A5 knockdown siRNAs (Fig. 6A) resulted in significant percentage reductions of 22 and 30.5% (siRNA 185 and 492, respectively; p < 0.0001) of total cellular melanin compared with the control cells, demonstrating that SLC24A5 expression plays an important role in melanin synthesis in these cells. The effect of SLC24A5 knockdown on melanogenesis was even clearer when assessed in depigmented melanocytes (Fig. 6B). In these cultures the difficulty of measuring a reduction in de novo melanin synthesis against a large background of pre-existing melanin was substantially lessened. The pigment content of NHM cultured in melanoblast media for 1 week was greatly reduced (Fig. 6B, MB), and switching these cells back into normal melanocyte media restored pigment production after 5 days (Fig. 6B, MGM). Transfection of de-differentiated cells with SLC24A5 siRNA knockdown duplexes (185 and 492) almost completely blocked pigment synthesis after switching back into melanocyte media compared with the control transfection (Fig. 6B, SC) with a normalized percentage difference in melanin content of 80-90% (p < 0.0001). These results demonstrate for the first time that SLC24A5 expression is necessary for melanin production in human epidermal melanoblasts stimulated to differentiate. Melanosomal Markers Are Down-regulated after SLC24A5 Knockdown—Protein extracts prepared from re-pigmented melanoblasts treated with control or SLC24A5 knockdown siRNAs were probed by Western blotting for known melanosomal markers (Fig. 7). The expression of early melanosome markers pmel17 (85-kDa form) and MART1 and late melanosomal markers TYR and TYRP1 was substantially reduced after SLC245A5 knockdown. Conversely, expression of the lysosomal marker LAMP-1 was up-regulated after SLC24A5 knockdown, suggesting that SLC24A5 plays a role very early in melanosome biogenesis and that this process is intimately linked with lysosome biogenesis. Switching Threonine for Alanine at Residue 111 of NCKX5 or Residue 177 of NCKX2 Reduces Exchange Activity—The NCKX proteins have a high degree of amino acid sequence similarity in two regions that have been identified as essential for exchange activity (denoted α1 and α2). The non-synonymous SNP (rs1426654) that is associated with natural skin color variation lies within the first of these highly conserved regions. All NCKX proteins and all cross-species orthologues of NCKX5 have an alanine at this position, except the NCKX5 variant identified in light-skinned humans, which has a threonine (Fig. 8, A and B). Consistent with the notion that native NCKX5 is localized to an intracellular membrane, no NCKX activity could be measured across the plasma membranes of B16 or NHM cells using established methods (12Altimimi H.F. Schnetkamp P.P.M. Channels. 2007; 1: 62-69Crossref PubMed Scopus (51) Google Scholar) and neither was trafficking to the plasma membrane observed when Myc-tagged NCKX5 was expressed in HEK293 cells. Therefore, we were also unable to use current methodology (12Altimimi H.F. Schnetkamp P.P.M. Channels. 2007; 1: 62-69Crossref PubMed Scopus (51) Google Scholar, 15Kang K.J. Kinjo T.G. Szerencsei R.T. Schnetkamp P.P. J. Biol. Chem. 2005; 280: 6823-6833Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) to measure the NCKX activity of NCKX5 in the heterologous system used successfully for the NCKX2 isoform. In contrast, some trafficking of Myc-tagged NCKX5 to the plasma membrane was observed in a High Five insect cell express
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