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• W2023388715 abstract "The Long QT Syndrome is a cardiac disorder associated with ventricular arrhythmias that can lead to syncope and sudden death. One prominent form of the Long QT syndrome has been linked to mutations in the HERG gene (KCNH2) that encodes the voltage-dependent delayed rectifier potassium channel (IKr). In order to search for HERG-interacting proteins important for HERG maturation and trafficking, we conducted a proteomics screen using myc-tagged HERG transfected into cardiac (HL-1) and non-cardiac (human embryonic kidney 293) cell lines. A partial list of putative HERG-interacting proteins includes several known components of the cytosolic chaperone system, including Hsc70 (70-kDa heat shock cognate protein), Hsp90 (90-kDa heat shock protein), Hdj-2, Hop (Hsp-organizing protein), and Bag-2 (BCL-associated athanogene 2). In addition, two membrane-integrated proteins were identified, calnexin and FKBP38 (38-kDa FK506-binding protein, FKBP8). We show that FKBP38 immunoprecipitates and co-localizes with HERG in our cellular system. Importantly, small interfering RNA knock down of FKBP38 causes a reduction of HERG trafficking, and overexpression of FKBP38 is able to partially rescue the LQT2 trafficking mutant F805C. We propose that FKBP38 is a co-chaperone of HERG and contributes via the Hsc70/Hsp90 chaperone system to the trafficking of wild type and mutant HERG potassium channels. The Long QT Syndrome is a cardiac disorder associated with ventricular arrhythmias that can lead to syncope and sudden death. One prominent form of the Long QT syndrome has been linked to mutations in the HERG gene (KCNH2) that encodes the voltage-dependent delayed rectifier potassium channel (IKr). In order to search for HERG-interacting proteins important for HERG maturation and trafficking, we conducted a proteomics screen using myc-tagged HERG transfected into cardiac (HL-1) and non-cardiac (human embryonic kidney 293) cell lines. A partial list of putative HERG-interacting proteins includes several known components of the cytosolic chaperone system, including Hsc70 (70-kDa heat shock cognate protein), Hsp90 (90-kDa heat shock protein), Hdj-2, Hop (Hsp-organizing protein), and Bag-2 (BCL-associated athanogene 2). In addition, two membrane-integrated proteins were identified, calnexin and FKBP38 (38-kDa FK506-binding protein, FKBP8). We show that FKBP38 immunoprecipitates and co-localizes with HERG in our cellular system. Importantly, small interfering RNA knock down of FKBP38 causes a reduction of HERG trafficking, and overexpression of FKBP38 is able to partially rescue the LQT2 trafficking mutant F805C. We propose that FKBP38 is a co-chaperone of HERG and contributes via the Hsc70/Hsp90 chaperone system to the trafficking of wild type and mutant HERG potassium channels. The Long QT Syndrome is a cardiac disorder characterized by a prolongation of the QT interval on the surface electrocardiogram that has been associated with ventricular arrhythmias whose clinical features range from minor dizziness to seizure, syncope, and sudden death. One prominent form of the Long QT syndrome (LQT2) 2The abbreviations used are: LQTS, long QT syndrome; HERG, human ether-a-go-go-related gene; WT, wild type; ER, endoplasmic reticulum; Hsc70, 70-kDa heat shock cognate protein; Hsp90, 90-kDa heat shock protein; CFTR, cystic fibrosis transmembrane conductance regulator; CHIP, C-terminal of Hsp70-interacting protein; Bag-2, BCL-associated athanogene 2; Hop, Hsp-organizing protein; FKBP38, 38-kDa FK506-binding protein; TPR, tetratricopeptide repeat; HA, hemagglutinin; HERG-C, C terminus of HERG; HEK, human embryonic kidney; PBS, phosphate-buffered solution; siRNA, small interfering RNA; AU, arbitrary units. is localized to chromosome 7 (1Curran M.E. Splawski I. Timothy K.W. Vincent G.M. Green E.D. Keating M.T. Cell. 1995; 80: 795-803Abstract Full Text PDF PubMed Scopus (2002) Google Scholar) and has been linked to genetic mutations in the KCNH2 gene that encodes the HERG subunit. The HERG channel is a tetramer of HERG subunits that comprises the α-subunit of the voltage-dependent delayed rectifier potassium current (IKr) (2Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2161) Google Scholar). To date, more than 200 naturally occurring LQT2 mutants have been identified that result in either abnormal HERG channel function or a decrease in cell surface localization. Approximately 13% of these HERG mutations are characterized as trafficking-deficient mutants and represent the most dominant mechanism for the loss of HERG function in LQT2 (3Anderson C.L. Delisle B.P. Anson B.D. Kilby J.A. Will M.L. Tester D.J. Gong Q. Zhou Z. Ackerman M.J. January C.T. Circulation. 2006; 113: 365-373Crossref PubMed Scopus (337) Google Scholar). HERG trafficking mutants represent a potential target for pharmacological intervention as roughly 70% can be rescued, suggesting that the folding defects might be subtle (3Anderson C.L. Delisle B.P. Anson B.D. Kilby J.A. Will M.L. Tester D.J. Gong Q. Zhou Z. Ackerman M.J. January C.T. Circulation. 2006; 113: 365-373Crossref PubMed Scopus (337) Google Scholar). Under physiological conditions wild type (WT) HERG exhibits two bands on Western blot analysis, with the generation of both bands involving asparagine (Asn)-linked glycosylation (4Zhou Z. Gong Q. Ye B. Fan Z. Makielski J.C. Robertson G.A. January C.T. Biophys. J. 1998; 74: 230-241Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). The core-glycosylated, immature form is represented by a 135-kDa band and is present in the endoplasmic reticulum (ER), whereas the fully glycosylated mature protein is observable as a 155-kDa band and represents HERG either in the Golgi apparatus or at the cell surface (5Petrecca K. Atanasiu R. Akhavan A. Shrier A. J. Physiol. 1999; 515 (, Pt. 1,): 41-48Crossref PubMed Scopus (127) Google Scholar, 6Gong Q. Anderson C.L. January C.T. Zhou Z. Am. J. Physiol. 2002; 283 (-H84): H77Crossref PubMed Scopus (20) Google Scholar). Comparison of the intensity of these two bands provides a useful assay of HERG trafficking. The prevailing view is that mutants with trafficking deficiencies are recognized and retained as misfolded proteins by the quality control machinery of the ER (7Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1681) Google Scholar). Recently, it has been reported that HERG forms complexes with the molecular chaperones Hsc70 (70-kDa heat shock cognate protein), Hsp90 (90-kDa heat shock protein) (8Ficker E. Dennis A.T. Wang L. Brown A.M. Circ. Res. 2003; 92 (-e100): e87Crossref PubMed Google Scholar), and calnexin (9Gong Q. Jones M.A. Zhou Z. J. Biol. Chem. 2006; 281: 4069-4074Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), which are known to be important for polypeptide folding, sorting, transport, and degradation (10McClellan A.J. Tam S. Kaganovich D. Frydman J. Nat. Cell Biol. 2005; 7: 736-741Crossref PubMed Scopus (228) Google Scholar, 11Young J.C. Agashe V.R. Siegers K. Hartl F.U. Nat. Rev. Mol. Cell Biol. 2004; 5: 781-791Crossref PubMed Scopus (944) Google Scholar). Ficker et al. (8Ficker E. Dennis A.T. Wang L. Brown A.M. Circ. Res. 2003; 92 (-e100): e87Crossref PubMed Google Scholar) demonstrated that two LQT2-trafficking mutants, R752W and G601S, also interacted with Hsc70 and Hsp90 when retained in the ER and these interactions were prolonged relative to WT HERG. The recovery of channel trafficking and function by temperature reduction or pharmacological stabilization was tightly coupled to the dissociation of channel-chaperone complexes (8Ficker E. Dennis A.T. Wang L. Brown A.M. Circ. Res. 2003; 92 (-e100): e87Crossref PubMed Google Scholar). This finding suggests that the Hsc70 and Hsp90 chaperone system is important for the folding of HERG and that regulation of exit from the ER is mechanistically linked to the dissociation of HERG from this complex. The Hsc70/Hsp90 network has been characterized more extensively for the cystic fibrosis transmembrane conductance regulator (CFTR) (12Wang X. Venable J. LaPointe P. Hutt D.M. Koulov A.V. Coppinger J. Gurkan C. Kellner W. Matteson J. Plutner H. Riordan J.R. Kelly J.W. Yates J.R. II I Balch W.E. Cell. 2006; 127: 803-815Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 13Riordan J.R. Annu. Rev. Physiol. 2005; 67: 701-718Crossref PubMed Scopus (193) Google Scholar) and the glucocorticoid receptor (14Hernandez M.P. Sullivan W.P. Toft D.O. J. Biol. Chem. 2002; 277: 38294-38304Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Thus far, interactions have been identified between CFTR and Hsc70 (15Yang Y. Janich S. Cohn J.A. Wilson J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9480-9484Crossref PubMed Scopus (282) Google Scholar), calnexin (16Pind S. Riordan J.R. Williams D.B. J. Biol. Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar), Hsp90 (17Loo M.A. Jensen T.J. Cui L. Hou Y-X. Chang X.-B Riordan J.R. EMBO J. 1998; 17: 6879-6887Crossref PubMed Scopus (299) Google Scholar), Hdj-2 (18Meacham G.C. Lu Z. King S. Sorscher E. Tousson A. Cyr D.M. EMBO J. 1999; 18: 1492-1505Crossref PubMed Scopus (272) Google Scholar, 19Youker R.T. Walsh P. Beilharz T. Lithgow T. Brodsky J.L. Mol. Biol. Cell. 2004; 15: 4787-4797Crossref PubMed Scopus (129) Google Scholar), C-terminal of Hsp70-interacting protein (CHIP) (20Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nat. Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (707) Google Scholar, 21Younger J.M. Ren H-Y. Chen L. Fan C.-Y. Fields A. Patterson C. Cyr D.M. J. Cell Biol. 2004; 167: 1075-1085Crossref PubMed Scopus (145) Google Scholar, 22Arndt V. Daniel C. Nastainczyk W. Alberti S. Hohfeld J. Mol. Biol. Cell. 2005; 16: 5891-5900Crossref PubMed Scopus (157) Google Scholar), and Bcl-2-associated athanogene 2 (BAG-2) (22Arndt V. Daniel C. Nastainczyk W. Alberti S. Hohfeld J. Mol. Biol. Cell. 2005; 16: 5891-5900Crossref PubMed Scopus (157) Google Scholar). However, little is known about the protein complexes that form between HERG and molecular chaperones to facilitate HERG folding, maturation, and retention. To identify additional possible HERG-interacting chaperones we conducted a proteomic analysis. This revealed several known cytosolic chaperones, including Hsc70, Hsp90, Hdj-2, Hsp-organizing protein (Hop), and Bag-2 as well as two transmembrane proteins that included calnexin and FKBP38 (38-kDa FK506-binding protein, FKBP8). FKBP38 is of particular interest in that it is almost entirely exposed to the cytosol and is related to the known tetratricopeptide repeat (TPR) domain co-chaperone FKBP52 (23Peattie D.A. Harding M.W. Fleming M.A. DeCenzo M.T. Lippke J.A. Livingston D.J. Benasutti M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10974-10978Crossref PubMed Scopus (231) Google Scholar). It contains an N-terminal region of unknown function, an FK506 binding peptidylprolyl cis/trans isomerase domain, a TPR domain predicted to interact with Hsc70 and/or Hsp90, a calmodulin binding motif, and a C-terminal transmembrane anchor (24Lam E. Martin M. Wiederrecht G. Gene. 1995; 160: 297-302Crossref PubMed Scopus (70) Google Scholar). The TPR domain of FKBP38 is closely related to the TPR domain present in co-chaperones of Hsc70 and Hsp90. Some co-chaperone TPR domains, such as those in Hop, specifically distinguish between Hsc70 and Hsp90; others, such as that in CHIP, can bind either Hsc70 or Hsp90 (11Young J.C. Agashe V.R. Siegers K. Hartl F.U. Nat. Rev. Mol. Cell Biol. 2004; 5: 781-791Crossref PubMed Scopus (944) Google Scholar). The structural basis of Hsc70 and Hsp90 binding by TPR domains is known (25Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar), and the residues required are absolutely conserved in FKBP38. We hypothesize that as a membrane protein FKBP38 may provide a direct link between the cytosolic chaperones and ER retention or export mechanisms of HERG. Here we characterize the biochemical and functional effect of FKBP38 on WT HERG and a HERG trafficking mutant. Our results indicate that FKBP38 is important for the trafficking of WT HERG and is capable of rescuing the LQT2 HERG trafficking mutant F805C. Preparation of cDNA Constructs—The generation of N-terminal Myc-tagged and N-terminal hemagglutinin (HA)-tagged HERG has been described previously (26Akhavan A. Atanasiu R. Shrier A. J. Biol. Chem. 2003; 278: 40105-40112Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The HERG missense mutant F805C was engineered using the QuikChange XL site-directed mutagenesis kit (Stratagene) and a HERG-C cassette as the PCR template as described previously (26Akhavan A. Atanasiu R. Shrier A. J. Biol. Chem. 2003; 278: 40105-40112Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The cDNA of HA-tagged FKBP38 was kindly provided by Dr. Nakayama (Fukuoka, Japan) (27Shirane M. Nakayama K.I. Nat. Cell Biol. 2003; 5: 28-37Crossref PubMed Scopus (258) Google Scholar). Cell Culture and Transfection—HEK-293 and HL-1 cells were used and maintained as previously described (26Akhavan A. Atanasiu R. Shrier A. J. Biol. Chem. 2003; 278: 40105-40112Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). HEK-293 cells were cultured in α-minimal essential medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were cultured at 37 °C in 5% CO2. Transfections were carried out using either Lipofectamine or Lipofectamine 2000 as described by the manufacturer. HEK-293 cells were not used beyond 30 passages. Stable Cell Line Generation—A stable cell line for WT HERG was generated using the G-418 selection method. Briefly, transfected cells were maintained in α-minimal essential medium supplemented with 800 μg/ml of G-418 (Invitrogen) for 10 to 15 days. G-418-resistant colonies were selected by their protein expression levels and purity as determined by Western blot analysis and immunocytochemistry. Mass Spectrometry Analysis and Data Base Mining—Four proteomic screens of immunoprecipitated Myc-tagged HERG transfected into cardiac (HL-1) (28Claycomb W.C. Lanson Jr., N.A. Stallworth B.S. Egeland D.B. Delcarpio J.B. Bahinski A. Izzo Jr., N.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2979-2984Crossref PubMed Scopus (1250) Google Scholar, 29White S.M. Constantin P.E. Claycomb W.C. Am. J. Physiol. 2004; 286 (-H829): H823Crossref PubMed Scopus (336) Google Scholar) and non-cardiac (HEK-293) cell lines were performed. The Myc-immunoprecipitated HERG-bound complexes were separated by SDS-PAGE and stained by Coomassie blue. Individual bands were destained, excised, reduced, sulfur-alkylated, and digested with trypsin using a robotic digester (Micromass) at the Protein Unit Facility of the Genome Quebec Proteomics Platform. The resultant peptide mixtures were analyzed by direct on-line liquid chromatography tandem mass spectrometry (LC-MS/MS). The MS/MS spectra were evaluated using Mascot software (Matrix Science) to identify tryptic peptide sequences matched to the National Center for Biotechnology Information (NCBI) non-redundant protein and nucleotide data bases (dbEST) with a confidence level of 95% or greater (30Perkins D.N. Pappin D.J.C. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6814) Google Scholar). Peptide sequence tags were generated from the MS/MS spectra as described (31Mann M. Wilm M. Anal. Chem. 1994; 66: 4390-4399Crossref PubMed Scopus (1318) Google Scholar). The location and function of each identified protein were assigned using NCBI, Swiss-Prot, InterProHome, and InterProScan data bases. Immunoprecipitation and Western Blotting—Twenty four hours post-transfection, cells were washed two times with cold PBS and then incubated in lysis buffer (0.5% Nonidet P-40, 75 mm NaCl, and 50 mm Tris, pH 8) plus a protease inhibitor mixture (Roche Applied Science) for a minimum of 15 min. Cells were homogenized by pipetting, harvested, and then left on ice for an additional 15 min with occasional vortexing. Detergent-insoluble material was sedimented at 16,000 × g for 30 min after which the resulting supernatant was collected and the protein concentration was determined with a detergent-compatible assay according to the manufacturer’s instructions (Bio-Rad). For immunoprecipitation, samples of 0.5 to 1 mg of protein were incubated in a volume of 0.5 to 1 ml and incubated overnight at 4 °C with either monoclonal mouse α-Myc (Santa Cruz Biotechnology, Inc.) (1:100), monoclonal mouse α-HA (Covance) (1:100), or polyclonal rabbit α-FKBP38 (1:200) provided by Dr. Nakayama (Fukuoka, Japan). The immunoprecipitation samples were incubated with Protein A-Sepharose beads for 2 h after which the beads were washed extensively and then resuspended in sample buffer 2× (12% 0.5 m Tris, pH 6.8, 5% β mercaptoethanol, 20% glycerol, 20% SDS, Bromphenol blue). Samples were resolved on a 7.5% polyacrylamide SDS gel and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked for 1 h with 5% nonfat dry milk and 0.1% Tween 20 in PBS and then incubated with the appropriate primary antibody for 1 h at room temperature, washed extensively, and then incubated with goat α-mouse/rabbit IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories). After extensive washing the membranes were visualized on x-ray films using the ECL Plus detection kit (Amersham Biosciences). Overexpression and siRNA—For all overexpression experiments, HEK-293 cells grown on 35-mm dishes were transiently transfected at 70-80% confluence with WT or F805C HERG and varying amounts of HA-FKBP38 (0, 0.2, or 0.4 μg) and an appropriate amount of empty vector to equalize total amount of transfected cDNA. After 48 h, cells were lysed as described above and equal amounts of protein were loaded and subjected to SDS-PAGE and Western blot analysis. For siRNA experiments, siRNA oligonucleotides targeting the 5′-AAGAGUGGCUGGACAUUCUGG-3′ sequence in the open reading frame of human FKBP38 mRNA and a control double-stranded RNA with a corresponding scrambled sequence (5′-AAGCGCGCUUUGUAGGAUUC-3′) were obtained from Dharmacon Research (Lafyette, CO) based on Ref. 27Shirane M. Nakayama K.I. Nat. Cell Biol. 2003; 5: 28-37Crossref PubMed Scopus (258) Google Scholar. HEK-293 cells stably expressing Myc-HERG-WT were seeded at low confluence (20-30%) on 35-mm dishes and transfected with siRNA oligonucleotides according to the manufacturer’s instructions using Lipofectamine and Lipofectamine Plus Reagent (Invitrogen). Cells were lysed 2, 4, or 6 days post-transfection and subjected to SDS-PAGE and Western blot analysis as described above. Immunolocalization—HEK-293 cells stably expressing WT HERG-Myc were seeded onto gelatin/fibronectin-coated coverslips. 24 h post-seeding, cells were washed with PBS and then fixed for 15 min with 4% formaldehyde at room temperature on an orbital shaker. Cells were then washed with PBS and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Nonspecific antibody binding was blocked by PBS containing 10% goat serum for 30 min at room temperature after which cells were incubated with blocking solution containing either α-Myc (1:1000), α-KDEL (1:600), or α-FKBP38 (1:1000) antibody for 1 h. Coverslips were extensively washed before being incubated with blocking solution containing either Alexa Fluor 488-conjugated mouse secondary antibody or Alexa Fluor 633-conjugated rabbit secondary antibody at 1:400. After washing with PBS the coverslips were mounted onto glass slides using Immuno-Fluore Mounting Medium (ICN Biomedicals). Images were acquired with a Zeiss Axiovert 200 automated inverted microscope. Densitometry and Statistical Analysis—Densitometric analysis was carried out using the digital imaging program ImageJ (National Institutes of Health). Each band was quantified by a mean pixel value after subtraction of background. Pixel values for the upper and lower HERG bands and the FKBP38 band were normalized to the loading control protein PP2A. For both the siRNA and overexpression experiments, HERG trafficking efficiency is represented by the ratio of the normalized HERG upper band to the total normalized HERG intensity (HERG upper + HERG lower). All experiments were repeated in triplicate. Paired t tests were used to compare groups of data to determine significant. HERG Interacts with Cytosolic Chaperones—To search for other possible chaperones or co-chaperones that interact with HERG we conducted a total of four proteomic screens of immunoprecipitated Myc-tagged HERG transfected into cardiac (HL-1) (28Claycomb W.C. Lanson Jr., N.A. Stallworth B.S. Egeland D.B. Delcarpio J.B. Bahinski A. Izzo Jr., N.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2979-2984Crossref PubMed Scopus (1250) Google Scholar, 29White S.M. Constantin P.E. Claycomb W.C. Am. J. Physiol. 2004; 286 (-H829): H823Crossref PubMed Scopus (336) Google Scholar) and non-cardiac (HEK-293) cell lines. The Myc-immunoprecipitated HERG-bound complexes were separated by SDS-PAGE, excised, subjected to tryptic digestion, and then analyzed by tandem mass spectrometry at the Montreal Genome Centre. Identified HERG-interacting chaperones and co-chaperones include Hsc70, Hsp90, Hdj2, Hop, Bag-2, and calnexin (Table 1). In addition to these abundant cytosolic chaperones we identified the immunophilin FKBP38, a putative co-chaperone that may interact with the Hsc70/Hsp90 chaperone network involved in the HERG processing pathway.TABLE 1Identified HERG-interacting chaperones and co-chaperonesProteinOther namesSpeciesaHuman protein was found in immunoprecipitations conducted in HEK-293 cells. Mouse protein was found in immunoprecipitations conducted in HL-1 cells.Accession number (NCBI)LocalizationDomainsHdj-2DJA1HumanNP_001530CytosolJ domainHsc7070-kDa heat shock cognate proteinHumanNP_006588CytosolMouseP63017Hsp9090-kDa heat shock proteinHumanNP_001017963CytosolMouseNP_034610HOPSTIP1; Hsp-organizing proteinHumanNP_006810Cytosol2 TPR domainsBAG-2BCL2-associated athanogene 2HumanNP_004273CytosolBAG domainFKBP3838-kDa FK506-binding protein, FKBP8HumanQ14318ER membranePPIbPPI, peptidylprolyl cis/trans isomerase., TPR, calmodulin binding motifCalnexinMajor histocompatibility complex class I antigen-binding protein p88HumanAAH42843ER membraneCalreticulin domain, calcium binding domaina Human protein was found in immunoprecipitations conducted in HEK-293 cells. Mouse protein was found in immunoprecipitations conducted in HL-1 cells.b PPI, peptidylprolyl cis/trans isomerase. Open table in a new tab HERG and FKBP38 Co-immunoprecipitate and Co-localize—Although FKBP38 is ubiquitously expressed, it is found in greatest abundance in the brain and the heart (24Lam E. Martin M. Wiederrecht G. Gene. 1995; 160: 297-302Crossref PubMed Scopus (70) Google Scholar). To confirm that there is an interaction between HERG and FKBP38 we conducted co-immunoprecipitation experiments. First, HEK-293 cells were transiently transfected with HA-tagged WT HERG and then lysed and immunoprecipitated with an antibody against endogenous FKBP38. As shown in Fig. 1A, both the mature and immature forms of HERG were immunoprecipitated with the α-FKBP38 antibody. It remains unclear whether FKBP38 truly interacts with both forms of the HERG protein, given that most chaperones release their substrate once it has reached its native state. It seems conceivable that FKBP38 is bound to HERG even after its release from the ER. This might be expected if FKBP38 is involved in a late HERG folding stage or in the ER exit of HERG. Second, we transiently transfected HEK-293 cells with HA vector alone, HA-tagged WT HERG, Myc vector alone, or Myc-tagged WT HERG and then lysed and immunoprecipitated with either α-HA or α-Myc antibody. Fig. 1B shows the presence of a band at ∼62 kDa that corresponds to FKBP38, indicating that endogenous FKBP38 is immunoprecipitated with both Myc-tagged and HA-tagged WT HERG but not with the vectors alone. Finally, immunoprecipitation studies were conducted in HEK-293 cells alone or stably expressing Myc-tagged WT HERG and in AP-1 cells stably expressing the HA-tagged sodium-hydrogen exchanger NheI. Fig. 1C shows that stably expressed Myc-tagged HERG can precipitate endogenous FKBP38 whereas the stably expressed HA-tagged sodium-hydrogen exchanger NheI cannot. Taken together these results indicate that HERG and FKBP38 do indeed interact and predict that the two proteins are present in the same cellular compartments. FKBP38 primarily localizes to the outer membrane of the mitochondria through its C-terminal membrane anchor where it is proposed to play a role in the regulation of apoptosis through its interaction with Bcl-2 (27Shirane M. Nakayama K.I. Nat. Cell Biol. 2003; 5: 28-37Crossref PubMed Scopus (258) Google Scholar). Recent immunofluorescence and cell fractionation data indicate that FKBP38 resides at both the ER and mitochondrial membranes (27Shirane M. Nakayama K.I. Nat. Cell Biol. 2003; 5: 28-37Crossref PubMed Scopus (258) Google Scholar, 32Edlich F. Weiwad M. Erdmann F. Fanghanel J. Jarczowski F. Rahfeld J-U. Fischer G. EMBO J. 2005; 24: 2688-2699Crossref PubMed Scopus (115) Google Scholar, 33Kang C.B. Tai J. Chia J. Yoon H.S. FEBS Lett. 2005; 579: 1469-1476Crossref PubMed Scopus (45) Google Scholar). To first confirm the ER localization of FKBP38 in our system we performed immunolocalization experiments in HEK-293 and HL-1 cells. As shown in Fig. 2 there is overlap between the ER marker (α-KDEL antibody) and FKBP38 indicating that FKBP38 is localized to the ER as well as the mitochondria. To determine whether HERG and FKBP38 co-localize in our cell system we co-stained HEK-293 cells stably expressing Myc-tagged HERG with an α-Myc antibody (mouse) and an α-FKBP38 antibody (rabbit). Fig. 3A displays the clear overlap between the two proteins indicating that they co-localize in HEK-293 cells. To determine whether these two proteins also co-localize in a more physiological system, we transfected HA-tagged HERG into a cardiac cell line (HL-1) and co-stained with an α-HA antibody for HERG and an α-FKBP38 antibody for endogenous FKBP38. Fig. 3B shows that similar to what was found in the HEK-293 cells, when the HL-1 cells were transfected with HERG there is co-localization of HERG and FKBP38 that appears as a perinuclear staining pattern (Fig. 3B). Taken together, these results show that FKBP38 is expressed in the ER where it co-localizes with HERG.FIGURE 3HERG co-localizes with FKBP38 in a cardiac (HL-1) and a non-cardiac (HEK-293) cell line. A, immunolocalization experiments were performed in HEK-293 cells stably expressing Myc-tagged HERG. HERG and FKBP38 were visualized using a monoclonal α-Myc and polyclonal α-FKBP38 primary antibody followed by Alexa488 (green)/Alexa633 (red)-conjugated secondary antibodies, respectively. B, HL-1 cells transfected with HA-tagged HERG were visualized as described for panel A, except that α-HA was used instead of α-Myc for HERG visualization. For panels A and B the third panel represents a merged image to demonstrate the overlap of the proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) FKBP38 Knock Down Reduces HERG Trafficking—To determine whether the interaction between HERG and FKBP38 has a role in the trafficking of HERG we used siRNA to reduce the level of FKBP38 expression in the HEK-293 cell model. Under control conditions, both the immature, core glycosylated ER form and the mature complex glycosylated form of HERG were observed by Western blot at days 2, 4, and 6 post-transfection. The targeted siRNA reduction of FKBP38 caused a marked diminution of FKBP38 expression by 2 days post-transfection whereas a double-stranded scrambled oligonucleotide had no effect on the expression level of FKBP38 (Fig. 4A). Importantly, this FKBP38 knock down on day 2 led to a statistically significant reduction in the HERG trafficking efficiency relative to control (HG upper/HG total) on day 4 (15.0 ± 1.8 relative to control 36.8 ± 8.4 arbitrary units (AU); p = 0.03) and day 6 (24.6 ± 1.24 relative to control 41.5 ± 5.5 AU; p = 0.02; Fig. 4B). We attribute the delay in the siRNA-induced FKBP38 reduction of HERG trafficking efficiency to the relative stabilities of FKBP38 and HERG. HERG is a stable protein with a half-life of ∼12-16 h at the cell surface; thus, a maximal diminution of mature HERG would not be expected on day 2 when FKBP38 levels are minimal but rather at the next time point, on day 4 as observed. Thus, the reduction of FKBP38 expression results in a decrease in the efficiency of HERG maturation. FKBP38 Rescues Trafficking of F805C—Given that siRNA knock down of FKBP38 reduced HERG maturation, we questioned whether overexpression of FKBP38 would augment HERG maturation and/or rescue a HERG trafficking mutant. To test these possibilities, we overexpressed HA-tagged FKBP38 in the presence of either WT HERG or the HERG trafficking mutant F805C. The F805C mutation is present in the cyclic nucleotide binding domain (34Delisle B.P. Anderson C.L. Balijepalli R.C. Anson B.D. Kamp T.J. January C.T. J. Biol. Chem. 2003; 278: 35749-35754Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and can be rescued by a reduction in temperature (35Ficker E. Obejero-Paz C.A. Zhao S. Brown A.M. J. Biol. Chem. 2002; 277: 4989-4998Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) or by the sarcoplasmic/endoplasmic reti" @default.
• W2023388715 created "2016-06-24" @default.
• W2023388715 creator A5013031320 @default.
• W2023388715 creator A5066943011 @default.
• W2023388715 creator A5073059790 @default.
• W2023388715 creator A5081149858 @default.
• W2023388715 date "2007-08-01" @default.
• W2023388715 modified "2023-10-16" @default.
• W2023388715 title "Co-chaperone FKBP38 Promotes HERG Trafficking" @default.
• W2023388715 cites W142850513 @default.
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