Huntingtin-interacting Proteins, HIP14 and HIP14L, Mediate Dual Functions, Palmitoyl Acyltransferase and Mg2+ Transport
2008; Elsevier BV; Volume: 283; Issue: 48 Linguagem: Inglês
10.1074/jbc.m801469200
ISSN1083-351X
AutoresAngela Goytain, Rochelle M. Hines, Gary A. Quamme,
Tópico(s)Ion channel regulation and function
ResumoPolyglutamine expansions of huntingtin protein are responsible for the Huntington neurological disorder. HIP14 protein has been shown to interact with huntingtin. HIP14 and a HIP14-like protein, HIP14L, with a 69% similarity reside in the Golgi and possess palmitoyl acyltransferase activity through innate cysteine-rich domains, DHHC. Here, we used microarray analysis to show that reduced extracellular magnesium concentration increases HIP14L mRNA suggesting a role in cellular magnesium metabolism. Because HIP14 was not on the microarray platform, we used real-time reverse transcriptase-PCR to show that HIP14 and HIP14L transcripts were up-regulated 3-fold with low magnesium. Western analysis with a specific HIP14 antibody also showed that endogenous HIP14 protein increased with diminished magnesium. Furthermore, we demonstrate that when expressed in Xenopus oocytes, HIP14 and HIP14L mediate Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with Michaelis constants of 0.87 ± 0.02 and 0.74 ± 0.07 mm, respectively. Diminished magnesium leads to an apparent increase in HIP14-green fluorescent protein and HIP14L-green fluorescent fusion proteins in the Golgi complex and subplasma membrane post-Golgi vesicles of transfected epithelial cells. We also show that inhibition of palmitoylation with 2-bromopalmitate, or deletion of the DHHC motif HIP14ΔDHHC, diminishes HIP14-mediated Mg2+ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14ΔDHHC mutant restored Mg2+ transport to values observed with wild-type HIP14. Although we did not directly measure palmitoylation of HIP14 in these studies, the data are consistent with a regulatory role of autopalmitoylation in HIP14-mediated Mg2+ transport. We conclude that the huntingtin interacting protein genes, HIP14 and HIP14L, encode Mg2+ transport proteins that are regulated by their innate palmitoyl acyltransferases thus fulfilling the characteristics of "chanzymes." Polyglutamine expansions of huntingtin protein are responsible for the Huntington neurological disorder. HIP14 protein has been shown to interact with huntingtin. HIP14 and a HIP14-like protein, HIP14L, with a 69% similarity reside in the Golgi and possess palmitoyl acyltransferase activity through innate cysteine-rich domains, DHHC. Here, we used microarray analysis to show that reduced extracellular magnesium concentration increases HIP14L mRNA suggesting a role in cellular magnesium metabolism. Because HIP14 was not on the microarray platform, we used real-time reverse transcriptase-PCR to show that HIP14 and HIP14L transcripts were up-regulated 3-fold with low magnesium. Western analysis with a specific HIP14 antibody also showed that endogenous HIP14 protein increased with diminished magnesium. Furthermore, we demonstrate that when expressed in Xenopus oocytes, HIP14 and HIP14L mediate Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with Michaelis constants of 0.87 ± 0.02 and 0.74 ± 0.07 mm, respectively. Diminished magnesium leads to an apparent increase in HIP14-green fluorescent protein and HIP14L-green fluorescent fusion proteins in the Golgi complex and subplasma membrane post-Golgi vesicles of transfected epithelial cells. We also show that inhibition of palmitoylation with 2-bromopalmitate, or deletion of the DHHC motif HIP14ΔDHHC, diminishes HIP14-mediated Mg2+ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14ΔDHHC mutant restored Mg2+ transport to values observed with wild-type HIP14. Although we did not directly measure palmitoylation of HIP14 in these studies, the data are consistent with a regulatory role of autopalmitoylation in HIP14-mediated Mg2+ transport. We conclude that the huntingtin interacting protein genes, HIP14 and HIP14L, encode Mg2+ transport proteins that are regulated by their innate palmitoyl acyltransferases thus fulfilling the characteristics of "chanzymes." Huntington disease is a progressive neurodegenerative disorder caused by an expansion of the CAG repeat in the huntingtin gene that confers an expanded polyglutamine (poly(Q)) stretch in the huntingtin protein (1Singaraja R.R. Hadano S. Metzler M. Givan S. Wellington C.L. Warby S. Yanai A. Gutekunst C.A. Leavitt B.R. Yi H. Fichter K. Gan L. McCutcheon K. Chopra V. Michel J. Hersch S.M. Ikeda J.E. Hayden M.R. Hum. Mol. Genet. 2002; 11: 2815-2828Crossref PubMed Scopus (174) Google Scholar). The function of the huntingtin protein is unclear but it interacts with many cytoskeletal and synaptic vesicle proteins that are essential for exocytosis and endocytosis (2Huang K. Yanai A. Kang R. Arstikaitis P. Singaraja R.R. Metzler M. Mullard A. Haigh B. Gauthier-Campbell C. Gutekunst C.A. Hayden M.R. El-Husseini A. Neuron. 2004; 44: 977-986Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 3DiFiglia M. Sapp E. Chase K. Schwarz C. Meloni A. Young C. Martin E. Vonsattel J.P. Carraway R. Reeves S.A. Boycea F.M. Aronin N. Neuron. 1995; 14: 1075-1081Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 4Velier J. Kim M. Schwarz C. Kim T.W. Sapp E. Chase K. Aronin N. DiFiglia M. Exp. Neurol. 1998; 152: 34-40Crossref PubMed Scopus (244) Google Scholar, 5Kegel K.B. Sapp E. Yoder J. Cuiffo B. Sobin L. Kim Y.J. Qin Z.H. Hayden M.R. Aronin N. Scott D.L. Isenberg G. Goldmann W.H. DiFiglia M. J. Biol. Chem. 2005; 280: 36464-36473Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). One of the interacting proteins identified by the yeast two-hybrid system is huntingtin-interacting protein 14, HIP14 (1Singaraja R.R. Hadano S. Metzler M. Givan S. Wellington C.L. Warby S. Yanai A. Gutekunst C.A. Leavitt B.R. Yi H. Fichter K. Gan L. McCutcheon K. Chopra V. Michel J. Hersch S.M. Ikeda J.E. Hayden M.R. Hum. Mol. Genet. 2002; 11: 2815-2828Crossref PubMed Scopus (174) Google Scholar). A related protein, HIP14-like (HIP14L), which has 69% homology to HIP14, was identified with an in silico data base search (1Singaraja R.R. Hadano S. Metzler M. Givan S. Wellington C.L. Warby S. Yanai A. Gutekunst C.A. Leavitt B.R. Yi H. Fichter K. Gan L. McCutcheon K. Chopra V. Michel J. Hersch S.M. Ikeda J.E. Hayden M.R. Hum. Mol. Genet. 2002; 11: 2815-2828Crossref PubMed Scopus (174) Google Scholar). As with huntingtin protein, HIP14 and HIP14L are evolutionary conserved and widely distributed among tissues. Whereas huntingtin is normally located on plasma and intracellular membranes and is associated with cytoplasmic vesicles and different organelles such as the Golgi, HIP14 appears to be primarily located in the Golgi and post-Golgi vesicles (2Huang K. Yanai A. Kang R. Arstikaitis P. Singaraja R.R. Metzler M. Mullard A. Haigh B. Gauthier-Campbell C. Gutekunst C.A. Hayden M.R. El-Husseini A. Neuron. 2004; 44: 977-986Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar).The functions of HIP14 are beginning to be clarified. The HIP14 secondary structure contains five predicted transmembrane domains that is reminiscent of a membrane receptor or transporter and possesses a cytoplasmic DHHC cysteine-rich domain defined by the Asp-His-His-Cys sequence motif (6Ducker C.E. Stettler E.M. French K.J. Upson J.J. Smith C.D. Oncogene. 2004; 23: 9230-9237Crossref PubMed Scopus (91) Google Scholar). The DHHC region confers palmitoyl acyltransferase activity giving it the ability to modify membranes by palmitoylation (6Ducker C.E. Stettler E.M. French K.J. Upson J.J. Smith C.D. Oncogene. 2004; 23: 9230-9237Crossref PubMed Scopus (91) Google Scholar, 7Linder M.E. Deschenes R.J. Nat. Rev. Mol. Cell. Biol. 2007; 8: 74-84Crossref PubMed Scopus (734) Google Scholar, 8El-Husseini A.E. Craven S.E. Chetkovich D.M. Firestein B.L. Schnell E. Aoki C. Bredt D.S. J. Cell Biol. 2000; 148: 159-172Crossref PubMed Scopus (237) Google Scholar). The presence of palmitate within the membrane protein affects how it interacts with lipid rafts and other membrane proteins (7Linder M.E. Deschenes R.J. Nat. Rev. Mol. Cell. Biol. 2007; 8: 74-84Crossref PubMed Scopus (734) Google Scholar). Palmitoylation by protein acyltransferases and depalmitoylation by acylprotein thioesterases regulate trafficking between membrane compartments and leading finally to protein degradation (7Linder M.E. Deschenes R.J. Nat. Rev. Mol. Cell. Biol. 2007; 8: 74-84Crossref PubMed Scopus (734) Google Scholar). Recently, Yanai et al. (9Yanai A. Huang K. Kang R. Singaraja R.R. Arstikaitis P. Gan L. Orban P.C. Mullard A. Cowan C.M. Raymond L.A. Drisdel R.C. Green W.N. Ravikumar B. Rubinsztein D.C. El-Husseini A. Hayden M.R. Nat. Neurosci. 2006; 9: 824-831Crossref PubMed Scopus (231) Google Scholar) reported that palmitoylation of huntingtin protein by HIP14 is important for its trafficking and function. Mutant huntingtin results in lower interaction with HIP14 and reduced palmitoylation that contribute to the formation of protein aggregates and enhanced neural toxicity.Magnesium is the second most abundant cation within the cell and plays an important role in many intracellular biochemical functions (10Quamme G.A. Kidney Int. 1997; 52: 1180-1195Abstract Full Text PDF PubMed Scopus (285) Google Scholar). Despite the abundance and importance of magnesium, little is known about how eucaryotic cells regulate their magnesium content. Intracellular free Mg2+ concentration is in the order of 0.5 mm that comprises 1–2% of the total cellular magnesium (11Dai L.-j. Ritchie G. Kerstan D. Kang H.S. Cole D.E.C. Quamme G.A. Physiol. Rev. 2001; 81: 51-84Crossref PubMed Scopus (263) Google Scholar). Accordingly, intracellular Mg2+ is maintained below the concentration predicted from the transmembrane electrochemical potential. Intracellular Mg2+ concentration is finely regulated likely by precise controls of Mg2+ entry, Mg2+ efflux, and intracellular Mg2+ storage compartments (11Dai L.-j. Ritchie G. Kerstan D. Kang H.S. Cole D.E.C. Quamme G.A. Physiol. Rev. 2001; 81: 51-84Crossref PubMed Scopus (263) Google Scholar). We have shown that Mg2+ entry is through specific and regulated magnesium pathways that are regulated by intrinsic mechanisms such that culture of cells in media containing low magnesium results in up-regulation of Mg2+ uptake into the cells. These data suggest that epithelial cells can sense the environmental magnesium and through transcription- and translation-dependent processes modulate Mg2+ transport and maintain magnesium balance.In an attempt to identify genes underlying cellular changes resulting from adaptation to low extracellular magnesium, we used oligonucleotide microarray analysis to screen for magnesium-regulated transcripts in epithelial cells (12Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar). One transcript, HIP14L, was significantly up-regulated by low extracellular magnesium suggesting that the synthesis was regulated by changes in cell magnesium. Real-time reverse transcriptase-PCR showed that both HIP14 and HIP14L transcripts and Western analysis showed that endogenous HIP14 protein was responsive to changes in magnesium concentration. As the predicted secondary structures of HIP14 and HIP14L amino acid sequences conformed to prototypic membrane transporters, the goal of the present study was to see if the encoded HIP14 and HIP14L proteins mediate Mg2+ transport. We used both electrophysiological and fluorescence studies to examine Mg2+ transport in HIP14- and HIP14L-expressing Xenopus laevis oocytes. Cellular distribution and subcellular localization of HIP14-GFP 2The abbreviations used are: GFPgreen fluorescent proteinMDCKMadin-Darby canine kidneyTRPMtransient receptor potential melastatinPBSphosphate-buffered salineTMDtransmembrane domainGAPDHglyceraldehyde-3-phosphate dehydrogenase. 2The abbreviations used are: GFPgreen fluorescent proteinMDCKMadin-Darby canine kidneyTRPMtransient receptor potential melastatinPBSphosphate-buffered salineTMDtransmembrane domainGAPDHglyceraldehyde-3-phosphate dehydrogenase. and HIP14L-GFP were determined by immunofluorescence microscopy in transfected MDCK and COS-7 cells. Furthermore, distribution of the fusion proteins were evaluated in response to changes in cellular magnesium. Our data indicates that HIP14 and HIP14L proteins mediate Mg2+ transport and the transcripts are regulated by magnesium, indicating that they might play a role in control of cellular magnesium homeostasis. Furthermore, HIP14-mediated Mg2+ transport is regulated by autopalmitoylation through its inherent palmitoyl acyltransferase activity making this an unique membrane transport system.MATERIALS AND METHODSOligonucleotide Microarray Analysis—Microarray analysis was performed according to the protocol recommended by Affymetrix (www.affymetric.com) using MG U74 Bv2 and MG U74 Cv2 arrays (Affymetrix, Santa Clara, CA) as described previously (12Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar). DNA fragments representing transcripts that were up-regulated with low magnesium were selected and prioritized according to properties characteristic of membrane transport proteins.Construction of Expression Vectors Encoding HIP14 and HIP14L—Mouse Hip14L cDNA was purchased from RIKEN number 2410004E01Rik. Human HIP14-GFP, hHIP14L, hHIP14ΔDHHC-GFP, and GODZ-FLAG constructs were gifts from Dr. Alaa El-Husseini (2Huang K. Yanai A. Kang R. Arstikaitis P. Singaraja R.R. Metzler M. Mullard A. Haigh B. Gauthier-Campbell C. Gutekunst C.A. Hayden M.R. El-Husseini A. Neuron. 2004; 44: 977-986Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). HIP14 and HIP14L constructs were in the pCI-neo vector.Sequence Analysis—The HIP14 and HIP14L cDNA sequences were determined by standard methods. The full-length amino acid sequences are in the GenBank™ data base (HIP14 accession human NP_056151, mouse NP_766142 and HIP14L, also termed HIP14-related or HIP14R, human BC056152, and mouse NM_028031). Protein motifs were identified using BLASTP and the SWISSPROT data base. Membrane topology was predicted by the SOSUI program based on Kyte-Doolittle hydrophobicity analysis.Quantitative Analysis of Hip14 and Hip14L Transcripts by Real-time Reverse Transcriptase-PCR—Total cell RNA was extracted by TRIzol (Invitrogen). Genomic DNA contamination was removed by the DNA-free™ kit (Ambion) prior to making first strand cDNA. Standard curves were constructed by serial dilution of a linear pGEM-T vector (Promega) containing the Hip14 and Hip14L genes. The primer set of mouse Hip14 was: forward, 5′-AGCATGCAGCGGGAGGAGG-3′ and reverse, 5′-CAATGGAGGAGGGTAACA-3′ and Hip14L was forward, 5′-CCGAAATGCTAAGGGAGAA-3′ and reverse, 5′-TCTCTGCTAGGGTGACGAT-3′. PCR products were quantified continuously with AB7000™ (Applied Biosystems) using SYBR Green™ fluorescence according to the manufacturer's instructions. The relative amounts of RNA were normalized to mouse β-actin transcripts.Western Blot Analysis of Endogenous Hip14 Protein—Polyclonal rabbit HIP14 antibody was generated by Singaraja et al. (1Singaraja R.R. Hadano S. Metzler M. Givan S. Wellington C.L. Warby S. Yanai A. Gutekunst C.A. Leavitt B.R. Yi H. Fichter K. Gan L. McCutcheon K. Chopra V. Michel J. Hersch S.M. Ikeda J.E. Hayden M.R. Hum. Mol. Genet. 2002; 11: 2815-2828Crossref PubMed Scopus (174) Google Scholar), commercialized and subsequently purchased from Sigma. Cells were suspended in lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 0.1% SDS) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin). The homogenates were pelleted at 1,000 rpm (75 × g) for 10 min and the supernatant and pellet fractions sampled. Protein concentrations were determined using the Bio-Rad protein assay reagent. SDS-PAGE was performed according to Laemmli (49Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206038) Google Scholar). For immunoblotting, the proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Hybond®, Amersham Biosciences) by semidry electroblotting for 80 min. Western analysis was performed by incubating the blots with anti-HIP14 antibody overnight at 4 °C followed by three washes with PBS, 0.1% Tween 20, 10 min each. The blots were then incubated with 1/5,000 horseradish peroxidase-conjugated donkey anti-rabbit secondary (Sigma) antibody for 1 h. After washing three times with PBS/Tween-20, 10 min each, the blots were visualized with ECL (Amersham Biosciences) according to the manufacturer's instructions. Hip14 protein was normalized to GAPDH prepared from the respective cell preparations. Cell preparations were incubated with mouse "α-GAPDH antibody (Sigma) diluted 1/5,000 in PBS, 1% BSA for 2 h and subsequently with horseradish peroxidase-conjugated goat secondary antibody at 1/10,000 in PBS, 1% BSA for 1 h to quantitate the GAPDH.Expression of Human HIP14 and HIP14L cRNAs in Xenopus Oocytes and Characterization of Mg2+ Transport—For Xenopus oocyte expression, cRNA was synthesized from hHIP14 and hHIP14L or mHip14L cDNA constructs, linearized and then transcribed with T7 polymerase in the presence of m7GpppG cap using the mMESSAGE MACHINE™ T7 Kit (Ambion) transcription system. Preparation of oocytes, injection with cRNA, and two-electrode voltage-clamp were as previously described (13Goytain A. Hines R. El-Husseini A. Quamme G.A. J. Biol. Chem. 2007; 282: 8060-8068Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Oocytes were studied 3–5 days following injection. Permeability ratios were calculated using the Nernst relation and apparent Km and Vmax values with Eadie-Hofstee analysis using non-linear regression analysis (12Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar).Epifluorescence microscopy was used to measure Mg2+ flux into single oocytes using the Mg2+-responsive mag-fura-2 fluorescence dye (13Goytain A. Hines R. El-Husseini A. Quamme G.A. J. Biol. Chem. 2007; 282: 8060-8068Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Oocytes were injected with 50 μm mag-fura-2 acid (Molecular Probes), 20 min prior to experimentation. The chamber (0.5 ml) was mounted on an inverted Nikon Diaphot-TMD microscope, with a Fluor ×10 objective, and a current (I)-voltage (V) plot determined. Subsequently, they were clamped at -70 mV for fluorescence measurements for the indicated times. Fluorescence was continuously recorded using a dual-excitation wavelength spectrofluorometer (Deltascan, Photon Technologies) with excitation for mag-fura-2 at 340 and 385 nm (chopper speed set at 100 Hz), and emission at 505 nm. Results are presented as the 340/385 ratio that reflects the intracellular Mg2+ concentration.Cell Culture—MDCK and COS-7 epithelial cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum, 110 mg/liter sodium pyruvate, 5 mm l-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin in a humidified environment of 5% CO2, 95% air at 37 °C. Where indicated, subconfluent cells were cultured in nominally Mg2+-free or normal 0.8 mm magnesium media (Stem Cell Technologies) for 12–16 h prior to harvest or processing for immunochemistry as previously described (13Goytain A. Hines R. El-Husseini A. Quamme G.A. J. Biol. Chem. 2007; 282: 8060-8068Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Other constituents of the Mg2+-free culture media were similar to the complete media.Immunofluorescence Confocal Microscopy—Subcellular localization of HIP14 and HIP14L were performed by immunofluorescence with transfection of tagged constructs. MDCK and COS-7 epithelial cells were transiently transfected with either pCI-HIP14-GFP or pCI-HIP14L-GFP using Lipofectamine 2000 (Invitrogen). Transfections were performed 8–9 h prior to culture in normal or low magnesium. Coverslips of cultured cells were fixed at room temperature for 10 min in 2% paraformaldehyde. Cells were washed three times with phosphate-buffered saline containing 0.3% Triton X-100 (PBST) before each antibody incubation. The following primary antibodies were used: GM130 (a cis-Golgi matrix protein) and Rab5 (GTP-binding protein) that were raised in mouse (BD Transduction Labs). Alexa 488- and Alexa 568-conjugated secondary antibodies were obtained from Molecular Probes. All antibody reactions were performed in blocking solution composed of 2% normal goat serum in PBST for 1.5 h at room temperature. Alexa 350-conjugated phalloidin (Molecular Probes) was used to stain for actin in the indicated experiments to aid in delimiting peripheral membrane ruffles. Following staining, coverslips were then mounted on slides with Fluoromount-G glycerol-based mounting media (Southern Biotechnology).Oocytes were mounted in OCT cryostat medium and flash frozen in isopentane cooled in liquid nitrogen. Ten-μm thick sections were cut through frozen oocytes and mounted directly onto superfrost plus slides (Fisher Scientific). Sections were fixed in -20 °C methanol and processed for immunohistochemistry using the GFP primary antibody and anti-rabbit Alexa 568 secondary antibody.All epithelial cell images were taken using a ×63 water lens affixed to a Zeiss LSM 510 Meta microscope and AxioVision (epifluorescent) or LSM 510 Meta (confocal) software. Cells were selected from 10–12 fields of view and used for assessment of co-localization of antibody staining. Oocytes images were taken using a ×20 dry objective affixed to a Zeiss LSM 510 Meta microscope and LSM Image software.RESULTSHIP14 and HIP14L Are Magnesium-responsive Genes—With the knowledge that differential gene expression is involved with selective control of epithelial cell magnesium conservation, our strategy was to use microarray analysis to identify cDNAs that were up-regulated with low magnesium (12Goytain A. Quamme G.A. BMC Genomics. 2005; 6: 48Crossref PubMed Scopus (135) Google Scholar). We used RNA from immortalized mouse distal convoluted tubule epithelial cells cultured in media containing normal magnesium concentration or nominally magnesium-free media for 5 h prior to RNA harvest. As our objective was to identify novel transport proteins, we prioritized the differentially expressed candidates according to the predicted structural properties reported for hypothetical transporters. One of the selected cDNA fragments identified by an increase in transcript was Hip14L. A BLAST search of the GenBank™ data base was performed and another member of this family, HIP14, was identified. As Hip14 was not on the mouse Affymetrix MG U74 Bv2 and MG U74 Cv2 arrays (Affymetrix) used at the time of our initial microarray analysis, we first showed that both Hip14 and Hip14L transcripts are regulated by magnesium using real-time reverse transcriptase-PCR. Hip14 and Hip14L mRNAs significantly (p < 0.001) increased 2.9 ± 0.3- and 3.0 ± 0.2-fold, respectively, in mouse distal convoluted tubule epithelial cells, n = 6 independent preparations, cultured in low magnesium compared with normal cells confirming that they are differentially regulated by magnesium (Fig. 1A). Consonant with the increase in transcript there was an increase in endogenous Hip14 protein as determined with Western blotting using a HIP14-specific antibody (1Singaraja R.R. Hadano S. Metzler M. Givan S. Wellington C.L. Warby S. Yanai A. Gutekunst C.A. Leavitt B.R. Yi H. Fichter K. Gan L. McCutcheon K. Chopra V. Michel J. Hersch S.M. Ikeda J.E. Hayden M.R. Hum. Mol. Genet. 2002; 11: 2815-2828Crossref PubMed Scopus (174) Google Scholar). It was evident from the Western blots that the Hip14 protein band density increased in MDCK cell cultures in low magnesium relative to normal cells. Mean density of the Hip14 protein increased 2.5 ± 0.5- and 4.5 ± 1.0-fold, respectively, in supernatant and membrane pellets (Fig. 1B). HIP14 increased 1.9 ± 0.3- and 5.4 ± 1.3-fold in COS-7 cells prepared under similar conditions (Fig. 1C).Hydrophobicity plot using the SOUSI program predicted a secondary amino acid structure for HIP14 with six predicted transmembrane domains (TMDs) (Fig. 1D). The structure was initially predicted to be a membrane receptor or transporter (1Singaraja R.R. Hadano S. Metzler M. Givan S. Wellington C.L. Warby S. Yanai A. Gutekunst C.A. Leavitt B.R. Yi H. Fichter K. Gan L. McCutcheon K. Chopra V. Michel J. Hersch S.M. Ikeda J.E. Hayden M.R. Hum. Mol. Genet. 2002; 11: 2815-2828Crossref PubMed Scopus (174) Google Scholar). The DHHC-cysteine-rich domain consensus sequence is located within the cytoplasmic region between TMD4 and TMD5. Human HIP14L has a 69% similarity to HIP14 and possesses seven predicted TMDs. The cysteine-rich domain is located between TMD5 and TMD6 (Fig. 3A). We speculate that the first transmembrane region is cleaved on formation of the mature protein so that the DQHC motif would be located in the same position as HIP14.FIGURE 3Characterization of HIP14L. A, predicted secondary structure of mouse HIP14L. HIP14L possesses a palmitoyl acyltransferase motif (DQHC). B, current-voltage (I–V) relationships obtained from linear voltage steps in the presence of Mg2+-free solutions or those containing the indicated concentrations of MgCl2. Oocytes were clamped as described in the legend to Fig. 1B. Shown are average I–V curves obtained from control H2O-injected (n = 3) or HIP14L-expressing (n > 3) oocytes. C, summary of concentration-dependent Mg2+-evoked currents in HIP14L-expressing oocytes using a holding potential of -125 mV. Mean ± S.E. values are those given in Fig. 2B. The Michaelis constant determined with non-linear regression analysis was 0.87 mm. D, Mg2+ flux into HIP14L-expressing oocytes. Mag-fura-2 fluorescence ratios were measured in control and HIP14L-expressing oocytes, at resting potentials, in solutions consisting of nominally magnesium-free solutions and then with 2.0 mm MgCl2 with interruption and subsequently voltage-clamped at a holding potential of -70 mV, where indicated. Where indicated, 75 μm 2-bromopalmitate (2BrP) was added 3 h prior to experimentation. E, HIP14L mediates Mn2+ transport in expressing oocytes. Oocytes were initially voltage-clamped at -70 mV in the presence of Mn2+, a cation that quenches mag-fura-2 fluorescence at 340 nm. Note, the intensity determined at 340 nm diminished in HIP14L expressing cells but not water-injected control oocytes. Results are mean of tracings performed with 3 different oocyte preparations. F, surface expression of HIP14L-GFP protein in X. laevis oocytes determined with immunofluorescence. Left panel, HIP14L-GFP-injected oocyte treated with GFP antibody showing intense surface staining. Right panel, control water-injected oocytes tested with GFP antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)HIP14 and HIP14L Mediate Mg2+ Transport in Expressing Xenopus Oocytes—To determine whether HIP14 and HIP14L encode functional Mg2+ transporters, we prepared the respective human and mouse cRNAs, injected it into Xenopus oocytes, and measured Mg2+-evoked currents using two-microelectrode voltage clamp analysis and Mg2+ flux using mag-fura-2 fluorescence methodologies (13Goytain A. Hines R. El-Husseini A. Quamme G.A. J. Biol. Chem. 2007; 282: 8060-8068Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The electrophysiological data gave evidence for a rheogenic process with inward currents in HIP14 cRNA-injected oocytes, whereas there were no appreciable currents in control H2O- or total poly(A)+ RNA-injected cells from the same batch of oocytes. Fig. 2A shows mean current-voltage (I–V) plots. There was a mean +28 mV shift in reversal potential with a decade increase in magnesium concentration that approximated the theoretical value predicted by the Nernst relationship. Similar findings were obtained with HIP14L-expressing oocytes (Fig. 3B). Expression of GODZ, a Golgi-specific DHHC zinc finger protein (14Uemura T. Mori H. Mishina M. Biochem. Biophys. Res. Commun. 2002; 296: 492-496Crossref PubMed Scopus (50) Google Scholar), did not elicit or stimulate Mg2+ transport in control oocytes arguing against the notion that palmitoylation stimulated an endogenous Mg2+ transporter (see data presented below). GODZ protein acyltransferase is implicated in palmitoylation and regulated trafficking of diverse intracellular receptors and transporters (15Fang C. Deng L. Keller C.A. Fukata M. Fukata Y. Chen G. Luscher B. J. Neurosci. 2006; 26: 12758-12768Crossref PubMed Scopus (136) Google Scholar, 16Greaves J. Chamberlain L.H. J. Cell Biol. 2007; 176: 249-254Crossref PubMed Scopus (190) Google Scholar). Among the inherent properties of all transporters is the property of substrate saturation. The Mg2+-evoked currents elicited by HIP14 and HIP14L were saturable (Figs. 2B and 3C) demonstrating Michaelis constants (Km) of 0.87 ± 0.02 and 0.74 ± 0.07 mm, respectively. The substrate affinities of both transporters were commensurate with the physiological concentration of intracellular ionized Mg2+ of about 0.5 mm (10Quamme G.A. Kidney Int. 1997; 52: 1180-1195Abstract Full Text PDF PubMed Scopus (285) Google Scholar). Mag-fura-2 fluorescence determinations confirmed that the observed currents were due to Mg2+ influx (Fig. 2C). External magnesium increased the emission ratio of 340/385 excitation following voltage-clamp at -70 mV in HIP14 cRNA-injected oocytes but not control water-injected cells. Similar findings were observed using HIP14L-expressing oocytes (Fig. 3D). The electrophysiological experiments demonstrated that Mn2+ elicited currents in HIP14- and HIP14L-expressing oocytes (supplemental Fig. S1A). This was also evident using fluorescence measurement (Figs. 2D and 3
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