Cell Culture, Constructs, and Transfections
HeLa cells were maintained in minimal essential medium (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) and 100 IU/ml penicillin and streptomycin (Mediatech, Herndon, VA) at 37°C in a 5% CO2
incubator. Knockdown of HeLa cells stably expressing GalNacT2-green fluorescent protein (GFP) (Storrie et al., 1998
) was performed with Oligofectamine (Invitrogen, Carlsbad, CA) as described previously, using 40 nM small interfering RNA (siRNA) (Kapetanovich et al., 2005
), with the exception that a single transfection was sufficient for knockdown. The siRNAs were synthesized using the siRNA construction kit from Ambion (Austin, TX). The two siRNAs (−1, −2) used against Yip1A were made against the following target sequences, respectively: AAGTTACAGCATCGATGATCA and AATGGTTTTTTGCCTTGCTTT. The siRNA target sequence for Sar1a was AACCACTCTTCTTCACATGCT and AAGAACTGACCATTGCTGGCA for Sar1b. The control siRNA used throughout this study targeted bovine p115 and does not affect p115 in HeLa cells as described previously (Puthenveedu and Linstedt, 2004
Transient cotransfection of HeLa cells with both plasmid DNA and siRNA was performed with Lipofectamine 2000 (Invitrogen) according to manufacturer's specifications by using 150 ng of DNA and 10 pmol of siRNA per 0.5 ml. Yip1A was cloned out of a HeLa cDNA library using polymerase chain reaction (PCR) and inserted into the pCS2-MT vector by using the EcoRI and XbaI sites. The FLAG-tagged Yip1A construct was generated by cutting out the myc-epitope using the BamHI and EcoRI sites, followed by the addition of a single FLAG epitope by using a PCR-based loop-in modification of the QuikChange protocol (Stratagene, La Jolla, CA). The Yip1A rescue construct was generated by introducing silent mutations into the siRNA-2 target sequence using QuikChange (AATGGTCTTCTGTCTTGCTTT). The E95K mutation in the rescue construct was also generated by QuikChange using the sequence GAGCCACCTTTATTAGAAAAGTTAGGTATCAATTTTGACCAC. The Myc-tagged Sec13 control construct was cloned by PCR amplification from HeLa cDNA and inserting the PCR product into the pCS2-MT vector by using BamHI and ClaI. The Myc-ts045 vesicular stomatitis virus glycoprotein (VSV-G) construct was cloned using PCR to amplify ts045 VSV-G and inserting the product into the pCS2-MT vector at the BamHI site. Myc-tagged DP1 and DP1L1 were generated by PCR amplification from HeLa cDNA and insertion into EcoRI and XbaI sites in pCS2-MT. Sec61γ-GFP (both monomeric [mGFP] and dimerizing [dGFP] forms) was kindly provided by Dr. E. Snapp (Albert Einstein University, Bronx, NY).
Antibodies and Other Reagents
Antibodies used include mouse monoclonal antibody (mAb) against protein disulfide isomerase (PDI) (Abcam, Cambridge, MA); a rabbit polyclonal antibody (pAb) against Calnexin (Abcam); a rabbit pAb against BiP (Abcam); a rabbit pAb against LCB3 (Cell Signaling Technology, Danvers, MA); a rabbit pAb against tubulin (Abcam); rabbit pAbs against Giantin, GM130, and GRASP65 (kindly provided by Dr. A. Linstedt, Carnegie Mellon University, Pittsburgh, PA); a mouse mAb against ER-to-Golgi intermediate compartment (ERGIC)-53 (kindly provided by Dr. H.-P. Hauri, Biozentrum, University of Basel, Basel, Switzerland), the M2 mouse mAb against the FLAG epitope (Sigma-Aldrich); and a mouse mAb against the Myc epitope from the 9E10 cell line. Fluorophore-conjugated secondary antibodies were from Zymed Laboratories (South San Francisco, CA)/Invitrogen. A rabbit pAb was raised against a 6His-Yip1A (amino acids [aa] 1–89) fusion protein as antigen (Covance Research Products, Princeton, NJ) and was affinity purified on Affi-Gel 15 beads (Bio-Rad Laboratories, West Grove, PA) coupled with a glutathione transferase (GST)-Yip1A N-terminal fusion protein. A rabbit pAb was raised against GST-Sec13 (Covance Research Products) and affinity purified on Affi-Gel 10 beads (Bio-Rad Laboratories) coupled with 6His-Sec13. H89 was from Toronto Research Chemicals (North York, ON, Canada), and brefeldin A (BFA) was from Sigma-Aldrich.
Immunofluorescence and Immunoblot Assays
siRNA transfections were analyzed 48–72 h after transfection as indicated. Immunofluorescence procedures were as described previously (Kapetanovich et al., 2005
), except that a 20-min ice-cold methanol fixation was substituted for paraformaldehyde. Immunoblotting using our affinity-purified Yip1A antibody, as well as rabbit pAbs against calnexin and tubulin (Abcam), was performed on siRNA-treated cells harvested from 60-mm dishes as described previously (Kapetanovich et al., 2005
Light Microscopy and Photobleaching Experiments
All static images with the exception of those in were obtained using a Yokagawa spinning disk confocal scranhead (PerkinElmer Life and Analytical Sciences, Boston, MA) mounted on an Axiovert 200 microscope (Carl Zeiss, Jena, Germany) with a 100× 1.4 numerical aperture (NA) objective (Carl Zeiss) and acquired using a 12-bit Orca ER digital camera (Hamamatsu Photonics, Hamamatsu City, Japan). Maximal value projections of sections at 0.3-μm spacing (4-6/cell) were acquired using Imaging Suite software (PerkinElmer Life and Analytical Sciences). Quantitation of COPII assembly was carried out using ImageJ (National Institutes of Health, Bethesda, MD) as described previously (Kapetanovich et al., 2005
). The wide field images in were obtained with a 63× 1.3 NA objective on an Axioplan microscope (Carl Zeiss) and acquired with QED software and a 12-bit Orca ER digital camera (Hamamatsu Photonics).
Figure 4. The whorled ER phenotype is rescued by a wild type but not mutant siRNA-immune Yip1A construct. Cells cotransfected with Yip1A siRNA-2 and either a control Sec13-Myc construct (A and B), a siRNA-immune wild-type FLAG-Yip1A construct (C and D), or a siRNA-immune (more ...)
Fluorescence recovery after photobleaching (FRAP) analyses were performed on a 510 Meta/UV Duoscan Spectral Confocal with LSM Zen 2007 software by using a 100× 1.4 NA objective. Regions of interest were subjected to photobleaching with repeated pulses at 100% laser power with the pinhole wide open to obtain maximal depth of field. Recovery after photobleaching was monitored with attenuated laser power. To generate the fluorescence recovery curves, fluorescence within the photobleached region of interest at each time point was first normalized to that of a nonbleached reference region to account for general loss of fluorescence due to image acquisition. Recovery curves were then generated by setting the fluorescence intensity before bleaching to 100% and the intensity after the last bleach pulse to 0%.
Thirty-five-millimeter dishes of HeLa cells treated with control and Yip1A siRNA were fixed 48 or 72 h after transfection with 2% glutaraldehyde/phosphate-buffered saline (PBS) for 30 min at room temperature. After three washes in PBS, cells were further fixed in 2% potassium permanganate/H2O for 45 min, washed three times in distilled H2O, followed by dehydration in an ascending series of ethanol (10–100%). Samples were then infiltrated in a 1:1 mixture of Epon-Araldite and 100% ethanol. After 30 min, the mixture was exchanged with 100% Epon-Araldite and held in a desiccator for 60 h. Samples were then transferred for 24 h each at 30, 40, 50, and 60°C. Epoxy disks were removed from the dishes, and areas of the disk with cells were cut out and glued onto a blank embedding capsule with two-part epoxy. Thin (100-nm) sections were cut using a DDK diamond knife on a Reichert-Jung Ultracut E ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained with lead citrate for 2 min. The grids were viewed on an H-7100 transmission electron microscope (Hitachi High Technologies America, Pleasanton, CA) operating at 75 kV. Digital images were obtained using an AMT Advantage 10 charge-coupled device Camera System (Advanced Microscopy Techniques, Danvers, MA) and ImageJ (National Institutes of Health).
For the pull-down of DP1 and DP1L1 with endogenous Yip1A, a 10-cm plate of HeLa cells was transfected with either Myc-DP1 or Myc-DP1L1 by using CaPO4. After 72 h, cells were washed with ice-cold PBS and then scraped and solubilized for 30 min at 4°C for in HKT lysis buffer (100 mM KCl, 1% Triton X-100, 20 mM KHEPES, pH 7.2, and protease inhibitors). The lysate was then passed five times through a 25-gauge needle and then centrifuged for 20 min at 14,000 × g. The lysates were then incubated with 5 μl of antibody (immunoglobulin [Ig]G or Yip1A) bound to 15 μl of protein A-Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) that had been washed in lysis buffer. After a 2-h incubation at 4°C, the beads were washed three times with lysis buffer. The beads were then boiled for 10 min in 50 μl of 2× reducing sample buffer and resolved on a 10% SDS gel. Immunoblotting was performed using anti-Myc antibodies.
For the pull-down of DP1 using the FLAG-Yip1A constructs, 2 × 10-cm plates of HeLa cells were transfected with either Myc-DP1 only, FLAG-Yip1A-wt and Myc-DP1, or FLAG-Yip1A-E95K and Myc-DP1 by using jetPEI (Polyplus-Transfection, New York, NY) according to manufacturer's specifications. The immunoprecipitation was performed as described above; however, the lysates were incubated with anti-FLAG M2 agarose beads (Sigma-Aldrich).
ts045-VSV-G Transport Assay
To assay ER export in Yip1A knockdown cells, HeLa cells were cotransfected with either Myc-ts045 VSV-G and a control siRNA, or Myc-ts045 VSV-G and Yip1A siRNA by using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were placed at 40°C, the nonpermissive temperature for ER export of ts045 VSVG, to accumulate VSVG in the ER. After a 24-h incubation at the nonpermissive temperature, cells were shifted to the permissive temperature of 32°C for varying times. To assay ER export in cells expressing GFP-Sec61γ, HeLa cells were cotransfected with Myc-ts045 VSV-G and either dGFP-Sec61γ or mGFP-Sec61γ. Twenty-four hours after transfection, cells were shifted to 40°C and processed for the trafficking assay as described just above.
In Vitro COPII Assembly Assay in Yip1A Knockdown Cells
Cells in a 10-cm dish were transfected with either control or Yip1A siRNA by using Oligofectamine (Invitrogen) and passaged onto coverslips after 24 h. After an additional 24 h, a second siRNA transfection was performed. Knockdown was monitored 48 h after the second transfection by immunostaining with Calnexin antibodies. At this time, the whorled ER phenotype was confirmed in >95% of cells. The COPII assembly assay was performed precisely as described previously (Lee and Linstedt, 2000
) 72 h after the second transfection.