Polyclonal antibodies against PDI and Hsp90 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), the polyclonal and monoclonal GFP antibodies and the monoclonal PDI antibody from Abcam (Cambridge, MA), the polyoclonal antibody against α1-antitrypsin and the mouse antibody against FLAG from Sigma (St. Louis, MO), the monoclonal CFTR (M3A7) antibody from Upstate Biotechnology (Lake Placid, NY), and the polyclonal calnexin antibody from StressGen (San Diego, CA). The CTA and CTB antibodies were provided by the W. Lencer (Harvard). The polyclonal and monoclonal ERp29 antibodies were generous gifts from S. Mkrtchian (Karolinska Intitutet). The polyclonal Derlin-1 and Sec61 beta antibodies were gifts from T. Rapoport (Harvard). The polyclonal Derlin-2 antibody and the HA-tagged Derlin-1 construct were gifts from Y. Ye (National Institutes of Health). Purified CT and CTB were purchased from Calbiochem/EMD Biosciences (San Diego, CA). The pcDNA3.1 containing YFP construct and the mAb against HA were gifts from K. Verhey (University of Michigan). The CFTR construct was a gift from R. Frizzell (University of Pittsburgh). The pcDNA3.1 construct containing the FLAG-tag was a gift from E. Wiertz (Leiden University Medical Center). A HeLa cell line stably expressing the NHK mutant of α1-proteinase inhibitor was a gift from C. Wojcik (Indiana University).
Construction of the Derlin-1-YFP, Derlin-2-YFP, Wild-type Mouse PDI FLAG-tagged, and the I272W Mouse PDI FLAG-tagged Constructs
The Derlin-1-yellow fluorescent protein (YFP) construct was generated by PCR amplification of the Derlin-1 coding sequence using a hemagglutinin (HA)-tagged Derlin-1 construct as the template (Ye et al., 2004
), while the Derlin-2-YFP construct was generated by PCR amplification of the Derlin-2 coding sequence using a HeLa cell cDNA library as the template. The respective PCR-amplified fragments were subsequently ligated into a pcDNA3.1 vector containing YFP, with the YFP attached to the C-terminus of the protein. Wild-type mouse PDI-FLAG was generated by PCR amplification of the PDI coding sequence using a plasmid containing mouse PDI as the template. The PCR product was ligated into pcDNA3.1 containing the FLAG tag. Mutagenesis to obtain the I272W PDI-FLAG construct was conducted using the Stratagene QuikChange II Site-directed Mutagenesis Kit (La Jolla, CA) and the wild-type PDI-FLAG plasmid as the template. The mutant PDI construct was confirmed by sequencing.
293T cells were incubated with CT (10 nM) in HBSS at 37°C for 45 or 90 min. For permeabilization, 2 × 106 cells were resuspended in 100 μl of 0.02% digitonin in HCN buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM CaCl2, and 10 mM N-ethyl maleimide [NEM], and protease inhibitors), incubated on ice for 10 min and centrifuged at 16,000 × g for 10 min. The supernatant was removed and the pellet washed with PBS and resuspended in 100 μl of the original buffer. Fractions were analyzed by nonreducing SDS-PAGE and immunoblot analysis.
YFP, Derlin-1-YFP, Derlin-2-YFP, CFTR, mouse wild-type PDI FLAG-tagged, or mouse I274W PDI FLAG-tagged was transfected into 30% confluent 293T cells in a 10-cm dish using the Effectene system (Qiagen, Chatsworth, CA), and the cells were grown for an additional 48 h before experimentation.
293T cells were incubated with or without CT (10 nM or 30 nm) or CTB (10 nM) for 90 min. Cells were harvested, lysed in the buffer containing KOAc (150 mM), Tris, pH 7.5 (30 mM), MgCl2 (4 mM), and NEM (10 mM) with either 1% Triton X-100 or 1% deoxyBigChap, and centrifuged at 16,000 × g for 10 min, and the supernatant was used for immunoprecipitation experiments. Coimmunoprecipitation experiments between PDI-FLAG and Derlin-1 were performed using a lysis buffer containing 1% Tween 20. Where indicated, polyclonal Derlin-1, polyclonal Derlin-2, monoclonal ERp29, polyclonal Sec61 beta, monoclonal GFP, or polyclonal calnexin antibodies were added to the lysate and incubated overnight at 4°C. The immune complex was captured by the addition of protein A agarose beads (Invitrogen, Carlsbad, CA), washed, and subjected to nonreducing SDS-PAGE followed by immunoblotting with the appropriate antibody.
Toxin Membrane Pelleting Assay
CTA subunit (160 nM) or CT (160 nM) was incubated with proteoliposomes, PDI (2.3 μM), and reduced glutathione (GSH; 3 mM), for 60 min at 37°C. Samples were sedimented for 20 min at 40,000 rpm in a tabletop ultracentrifuge using a TLA 100.4 rotor. The supernatant and pellet fractions were analyzed by SDS-PAGE followed by immunoblotting.
Proteinase K Digestion
293T cells nontransfected or overexpressing Derlin-1-YFP or Derlin-1-HA were incubated with 0.04% digitonin for 5 min at 4°C and centrifuged at 14,000 rpm for 10 min, and the pellet was resuspended in a buffer containing HEPES (50 mM, pH 7.4), KOAc (150 mM), sucrose (250 mM), and MgCl2 (4 mM). The samples were incubated with 0.1 or 1 mg/ml Proteinase K for 30 min at 4°C, subjected to SDS-PAGE, and immunoblotted with an antibody against Derlin-1, Derlin-2, GFP, or HA.
Pulse-Chase Analysis of CFTR
Cells were pretreated with CTB (100 nM) for 3 h before the experiment. Analysis of CFTR degradation in HEK293T cells followed a previously published protocol (Zhang et al., 2002
RNA Isolation and mRNA Analysis by Semiquantitative RT-PCR
RNA extractions were carried out with the RNeasy mini kit (Qiagen), according to the manufacturer's instructions. Cells, 1 × 106, were used for the experiment. Samples were applied to the QIA shredder homogenizers (Qiagen). RNA was subjected to the RNase-Free DNase Set (Qiagen) and reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Richmond, CA) according to the manufacturer's protocol. Two microliters of cDNA products were amplified using the Expand High Fidelity PCR system (Roche, Indianapolis, IN) in the MgCl2-free buffer in the presence of specific primers for CFTR or GAPDH used at a concentration of 0.1 μM each. MgCl2 was added to the reaction at a concentration of 1 mM. Reactions were carried out in the Eppendorf Mastercycler Personal. A first cycle of 10 min at 95°C, 45 s at 65°C, and 1 min at 72°C was followed by 45 s at 95°C, 45 s at 65°C, and 1 min at 72°C for 30 cycles. Each set of reactions contained an RNA-negative control to rule out genomic DNA contamination. The following primers were used: CFTR: 5′-CAGCTGGAGAGGAGGAAGGGAG-3′ and 5′ GAGGGTCTGCA GGCAGGCAGTG-3′; GAPDH: 5′-ACCACAGTCCATGCCATCACTGCC-3′ and 5′-TC CACCACCCTGTTGCTGTAGCC-3′. CFTR yielded an amplification product of 765 base pairs and GAPDH of 453 base pairs, which were resolved by an agarose gel.