We derived polyclonal antisera #272, #277, #274, and #255 from rabbits immunized with keyhole limpet hemocyanin (KLH)-conjugated synthetic peptides corresponding to mTMC1 amino acids 21–39 (EEDKLPRRESLRPKRKRTR), 53–72 (DEETRKAREKERRRRLRRGA), 216-236 (GSLPRKTVPRAEEASAANFGV), and 731-747 (MKQQALENKMRNKKMAA), respectively. We ordered peptides from Princeton BioMolecules (Langhorne, PA) and antibodies from Covance Research Products (Denver, PA). We purchased polyclonal anti-β-tubulin and monoclonal anti-PDI (Abcam, Cambridge, MA), monoclonal anti-α-tubulin (Molecular Probes, Carlsbad, CA), polyclonal anti-GRP94, monoclonal anti-KDEL (Stressgen, San Diego, CA). Monoclonal anti-hemagglutinin (HA) antibodies were from Abcam and polyclonal anti-HA antibodies were from Covance.
We PCR-amplified the full-length mouse Tmc1
open reading frame from a previously reported cDNA clone in pGEM T-easy (1
). Our sense (5’-GCT AGC ATG TTG CAA ATC CAA GTG-3’) and antisense (5’-GGA TCC CTG GCC ACC AGC AGC TGC-3’) amplification primers contained NheI and BamHI restriction sites, respectively, for subsequent cloning. We used site-directed mutagenesis (QuickChange, Stratagene, La Jolla, CA) to insert one HA epitope tag (YPYDVPDYA) (7
) per expression construct at each of seven sites. Each pair of 67-bp mutagenic primers contained 27 bp (5’-TAC CCA TAT GAC GTC CCG GAC TAC GCC-3’) encoding the HA tag, flanked by two 20-bp Tmc1
sequences encoding each side of the target insertion site. The HA tag was inserted between amino acids 237 and 238 (HA1), 327 and 328 (HA2), 402 and 403 (HA3), 510 and 511 (HA4), 568 and 569 (HA5), 616 and 617 (HA6), and 671 and 672 (HA7) (). Clones were sequenced, to verify correct insertion of the HA-tag sequence without unwanted mutagenic events, and digested with NheI and BamHI. The cDNA inserts were purified by 1% agarose gel electrophoresis and QIAquick Gel Extraction (QIAGEN, Valencia, CA) and subcloned into NheI- and BamHI-digested pcDNA3.1 (-) (Invitrogen, Carlsbad, CA).
Fig. 1 Potential models of membrane topology, immunogenic peptides and epitope tags of mTMC1. (A) Deduced amino acid sequence of mTMC1 and transmembrane domains predicted by TMHMM2.0 (red (17)), MEMSAT (blue (27)), PSORT II (orange (28)), and TopPred (green (more ...)
Tissue Culture and Transient Transfection
COS-7 and HeLa cells were grown in DMEM (Invitrogen) with 10% fetal bovine serum in an incubator at 37°C with 5% CO2. For immunofluorescence assays, cells were grown on glass coverslips in six-well plates (Corning Glass, Lowell, MA) and transfected with the expression construct using Lipofectamine 2000 (Invitrogen) for COS-7 cells or FuGENE HD (Roche, Indianapolis, IN) for HeLa cells. COS-7 and HeLa cells were incubated in the transfection mix in a 5% CO2 incubator at 37°C for 18–24 h. Cells were grown in T75 flasks (Corning Glass, Lowell, MA) and incubated with transfection mix for 18–24 h for protein extraction.
Transiently transfected cells were detached with PBS containing 1mM EDTA for 5–10 minutes at 37°C. All of the following procedures were done on ice or at 4°C. Cells were washed twice in PBS, resuspended in MB buffer (210mM mannitol, 70mM sucrose, 1mM EGTA, and 10mM Hepes pH 7.5, protease inhibitor cocktail III (Calbiochem, Gibbstown, NJ)), and incubated for 1 h. Cells were lysed by 20 passages through a 29G x 1/2” (0.34mmx13mm) needle on a 1ml syringe (Kendall, Tyco Healthcare Group, Mansfield, MA). Microscopic examination assured that cell lysis was 99% complete. The lysed cell suspension was centrifuged at 600g for 10 min to pellet the nuclei. The supernatant was regarded as a whole cell lysate. Microsomes were prepared by a 20-min centrifugation of the whole cell lysate at 6,800g to remove mitochondria. The resulting supernatant was centrifuged for 1 h at 100,000g to obtain a microsomal pellet, which was resuspended in MB buffer.
Western blot analysis
Whole cell extracts and microsomal preparations were denatured by boiling in NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA) for 5 min. Samples were separated by SDS-PAGE on 4–12% Bis-Tris gels using either MOPS or MES buffer (Invitrogen). Proteins were transferred to PVDF membranes (Millipore, Billerica, MA), blocked overnight with 5% nonfat dry milk in TBST (10mM Tris-HCL pH 7.5, 150 mM NaCl, and 0.05% Tween-20), and probed with either anti-TMC1 antibody #277 or polyclonal anti-HA antibody (both at 1:400 dilution). Proteins were detected by ECL using a 1:10,000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (Amersham, Piscataway, NJ) or with the Typhoon Trio Plus imaging system (GE Healthcare Life Sciences, Piscataway, NJ) using a 1:2,500 dilution of ECL Plex Cy5 fluorescent anti-rabbit secondary antibody (Amersham).
All procedures were performed at room temperature. Cells on coverslips were washed three times with PBS, fixed with 4% paraformaldehyde in PBS for 10 min, and washed again three times with PBS for 10 min each. The plasma membrane was selectively permeabilized with 5-μg/ml (0.0005%) digitonin (Sigma, St. Louis, MO) in incubation buffer (0.3M sucrose, 2.5mM MgCl2, 0.1M KCl, 1mM EDTA, 10mM Pipes, pH 6.8) for 4 min. Alternatively, permeabilization of the plasma and ER membranes was achieved by incubation with 0.25% Triton X-100 in PBS for 10 min. Immediately following permeabilization, cells were washed three times with PBS and blocked with 2% bovine serum albumin (Roche) and 5% goat serum (Invitrogen) in PBS for 30 min. Cells were then incubated for 1 h with primary antibodies: polyclonal antisera #272, #255, or #274, or a monoclonal antibody to hemagglutinin. Cells were co-stained with antibodies to cytosolic tubulin, and ER luminal proteins PDI or GRP94 or soluble ER resident proteins carrying the KDEL retention signal. We initially used an anti-PDI antibody to stain COS-7 cells, but it produced high background staining with HeLa cells. We also tried antibodies against KDEL and GRP94, and found that anti-GRP94 gave the best results with HeLa cells. Cells were washed three times with PBS and incubated with secondary antibodies (Alexa Fluor, Invitrogen) for 30 min. After three 15-min washes with PBS, we mounted the coverslips onto glass slides and added ProLong Gold antifade reagent (Invitrogen). We randomly chose approximately 20 to 30 different areas of each sample for visualization with a Zeiss LSM510 confocal microscope, 63× apochromat oil immersion phase-contrast objective, and Texas Red or FITC optics.