Yeast Strains and Constructs—The following yeast strains were used: BY4741 (MATa his3Δ1 leu2Δ met15Δ ura3Δ) and NDY257 (BY4741 rtn1::kanMX4 rtn2::kanMX4 yop1::kanMX) (6 (link)). Strains expressing GFP fusions to the chromosomal alleles of YOP1 and RTN1 were obtained from Invitrogen. The plasmid encoding Sec63-GFP (pJK59) has been previously described (12 (link)). To make the plasmid encoding Rtn1-GFP (pCV19), the SEC63 portion of pJK59 was removed by digestion with XbaI and XhoI. The RTN1 gene, including 400 bp upstream of the start site, was PCR-amplified from yeast chromosomal DNA and inserted into the same sites.
Mammalian Plasmid Constructs—HA-DP1 was described previously (6 (link)). HA-Rtn3c was cloned by PCR amplifying Rtn3c (NCBI accession number: BC036717) from mouse cDNA with primers containing an N-terminal HA tag and inserted into pcDNA3.1D (Invitrogen). For Rtn4a-GFP, human Rtn4a was PCR-amplified from Rtn4a-Myc (described in a previous study (6 (link))) and ligated into the pAcGFP-N1 backbone (Clontech) using the XhoI and KpnI restriction sites at the 5′ and 3′ ends, respectively. For GFP-Rtn3c, Rtn3c was PCR-amplified from HA-Rtn3c and ligated into the pAcGFP-C1 backbone (Clontech) using the XhoI and EcoRI restriction sites. To clone GFP-Rtn4HD, the region encoding amino acids 961–1192 was PCR-amplified from human Rtn4a-Myc and inserted into pAcGFP-C1 using the XhoI/EcoRI restriction sites. GFP-DP1 was subcloned by PCR-amplifying mouse DP1 from HA-DP1 (described in a previous study (6 (link))) and inserting into pAc-GFP C1 using SacI/BamHI restriction sites. For GFP-Climp63, Climp63 was PCR-amplified from mouse cDNA and cloned into pAcGFP-C1 using the XhoI/EcoRI sites. Climp63Δlum-GFP was cloned by PCR amplifying the region encoding amino acids 1–115 (as described in (13 (link))) from GFP-Climp63 and inserted into pAcGFP-N1 using XhoI/EcoRI restriction sites. LBR-GFP was PCR-amplified from plasmid containing human LBR (14 (link)) and cloned into pAcGFP-N1 using the XhoI/BamHI restriction sites. For GFP-Sec61β, human Sec61β was PCR-amplified from the pcDNA3.1/GFP-Sec61β construct described previously (6 (link)), and inserted into pAcGFP-C1 using the BglII/EcoRI restriction sites. RFP-Sec61β was subcloned from GFP-Sec61β using the same restriction sites as above and inserted into an mRFP1 vector (pEGFP-C1 vector backbone where pEGFP has been replaced with mRFP1).
Microscopy of Yeast—Yeast strains were grown in synthetic complete medium (0.67% yeast nitrogen base and 2% glucose) and imaged live at room temperature using an Olympus BX61 microscope, UPlanApo 100×/1.35 lens, QImaging Retiga EX camera, and IPlabs version 3.6.4 software.
Screen for Mutations in Yeast RTN1 That Affect Localization—Error-prone PCR on RTN1 was performed using the GeneMorphII Random Mutagenesis Kit (Stratagene). The product of this reaction and pJK59 cut with XbaI and XhoI were used to transform wild-type yeast. Transformants were visually screened for those that showed perinuclear GFP localization.
Tissue Culture, Indirect Immunofluorescence, and Confocal Microscopy of COS-7 Cells—Cells were grown at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and subcultured every 2–3 days. Transfection of DNA into cells was performed using Lipofectamine 2000 (Invitrogen). After 5 h of transfection, cells were split onto acid-washed No. 1 coverslips and allowed to spread for an additional 24–36 h before being processed for indirect immunofluorescence.
For immunofluorescence, transfected cells were fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min, washed twice, and permeabilized in 0.1% Triton X-100 (Pierce) in PBS for 5–15 min. Cells were washed twice again and then probed with primary antibodies for 45 min in PBS containing 1% calf serum, at the following concentrations: rat anti-HA antibody (Roche Applied Science) at 1:200 dilution; mouse anti-αtubulin (Sigma) at 1:500 dilution; and rabbit anti-calreticulin antibody (Abcam) at 1:500 dilution. Cells were washed three times in PBS, and then incubated with various fluorophore-conjugated secondary antibodies for an additional 45 min (Alexafluor 488 or 555 anti-mouse at 1:250 dilution, Alexafluor 647 anti-rabbit 1:500 dilution, and Alexafluor 488 anti-rat 1:200 dilution (all from Invitrogen)). Cells were then washed and mounted onto slides using Fluoromount-G mounting medium (Southern Biotech).
All imaging for indirect immunofluorescence was captured using a Yokogawa spinning disk confocal on a Nikon TE2000U inverted microscope with a 100× Plan Apo numerical aperture 1.4 objective lens, and acquired with a Hamamatsu ORCA ER cooled charge-coupled device camera using MetaMorph 7.0 software. For image presentation, brightness and contrast were adjusted across the entire image using Adobe Photoshop 7.0, and images were converted from 12 to 8 bits.
Transmission Electron Microscopy—COS-7 cells expressing GFP-Rtn4HD were sorted in a MoFlo cell sorter (Cytomation). The resulting cell pellet was fixed for 1 h in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 m sodium cacodylate buffer (pH 7.4), washed in 0.1 m cacodylate buffer, and postfixed with a mixture of 1% OsO4 and 1.5% KFeCN6 for 30 min. The pellet was then washed in water and stained in 1% aqueous uranyl acetate for 30 min followed by dehydration in grades of alcohol (50%, 70%, and 95%, 2 × 100%). Next, the pellet was infiltrated in a 1:1 mixture of propylene oxide and TAAB Peon (Maria Canada Inc.) for 2 h, placed in pure TAAB Epon in a silicon-embedding mold, and polymerized at 65 °C for 48 h. Ultrathin sections (∼60–80 nm) were cut on a Reichert Ultracut-S microtome, placed onto copper grids, and stained with 0.2% lead citrate. Specimens were examined on a Tecnai G Spirit BioTWIN transmission electron microscope, and images were acquired with a 2k AMT charge-coupled device camera.
Fluorescence Recovery after Photobleaching—Transfected COS-7 cells were imaged in phenol red-free HyQ DME (HyClone) supplemented with 25 mm Hepes, pH 7.4, and 1% fetal bovine serum. FRAP experiments were conducted on a Zeiss LSM 510 NLO laser scanning inverted microscope using a Plan-Neofluor 100×/1.3 oil objective with argon laser line 488 nm (optical slices <1.2 mm for COS-7 and 4.2 μm for yeast). Mammalian cell experiments were done at 37 °C using an objective heater (Bioptechs) and an enclosed stage incubator (Zeiss). LSM 510 software version 3.2 was used for image acquisition and analysis. Magnification, laser power, and detector gains were identical across samples.
For all mammalian experiments, COS-7 cells were treated with 0.5 μm nocodazole, and all data were collected during the first 5–30 min of nocodazole addition. For photobleaching all constructs, except for LBR-GFP, the tubular ER was magnified using the 3× zoom function so that individual tubules could be seen clearly. For LBR-GFP, the microscope was focused onto the bottom of the nuclear envelope. Images taken for 5-s prebleaching, whereupon a region of interest of 65 × 65 pixels was photobleached at 100% laser power. After the photobleaching, images were taken at 1-s intervals for 75–300 s. Yeast cells were treated similarly except that the region of interest was 17 × 17 pixels, and images were taken every 2–4 s at room temperature.
Raw data were quantitated using Zeiss LSM510Meta software. For analysis, the fluorescence intensity of three regions of interest was measured: the photobleached region (PR), a region outside of the cell to check for overall background fluorescence (BR), and a region within the cell that was not photobleached to check for overall photobleaching and fluorescence variation (CR), for the entire course of the experiment. Microsoft Excel was used to normalize the relative fluorescence intensity, I, for each individual FRAP experiment using Equation 1. For data presentation, the mean averages of the normalized data for each set of FRAP experiments were plotted using GraphPad Prism 5.0, and fluorescence recovery curves were shown for the first 80–140 s of each experiment. Estimated half-times of recovery and mobile fraction values were calculated using the standard Michaelis-Menten equation.
Sucrose Gradient Centrifugation—For yeast sucrose gradient analysis, crude membranes were isolated from yeast strains expressing GFP-fused proteins at endogenous levels as follows: 200 ml of culture were grown to OD ∼1, pelleted and then resuspended in TKMG lysis buffer (50 mm Tris, pH 7.0, 150 mm KCl, 2 mm MgCl2, 10% glycerol, 1 mm EDTA, 1 mm PMSF, 1 mm 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride), flash frozen in liquid nitrogen, and ground using a mortar and pestle. Cell debris was separated from the lysate by low speed centrifugation for 5 min at ∼2,000 × g. Membranes were then pelleted by ultracentrifugation for 15 min at 100,000 × g and solubilized in 200 μl of TKMG buffer containing 1% digitonin. Solubilized lysate was centrifuged for 10 min at 12,000 × g to separate out any remaining cell debris. 100-μl of lysate were run on 5–30% w/v sucrose gradients for 4 h at 166,000 × g at 25 °C on a Beckman TLS55 rotor. Twenty gradient fractions were collected from top to bottom and analyzed by SDS-PAGE and immunoblotting with anti-GFP antibody (Roche Applied Science). 50 mg of apoferritin, catalase, and aldolase was used as molecular weight standards.
Xenopus washed membrane fractions were prepared in MWB (50 mm Hepes, pH 7.5, 2.5 mm MgCl2, 250 mm sucrose, and 150 mm potassium acetate) as previously described (6 (link)), incubated for 60 min at 25 °C in MWB containing 200 mm KCl and 0.5 mm GTP, and then solubilized for 30 min at 25 °C with either 2% Nonidet P-40 or 1.25% digitonin. Samples were pelleted for 15 min at 12,000 rpm, and the soluble fraction was loaded onto a 10–30% w/v sucrose gradient made with MWB containing 200 mm KCl, 0.1 mm GTP, and either 0.1% Nonidet P-40 or 0.1% digitonin, respectively. The sucrose gradient was centrifuged for 3 h, 45 min at 55,000 rpm. Sixteen gradient fractions were collected and analyzed by SDS-PAGE and immunoblotted with antibody against Xenopus Rtn4 (described in a previous study (6 (link))).
For mammalian sucrose gradient analysis, COS-7 cells transiently transfected with HA-DP1 or GFP-Sec61β were harvested by scraping and then lysed and solubilized in HKME buffer (25 mm Hepes, pH 7.8, 150 mm potassium acetate, 2.5 mm magnesium acetate, 1 mm EDTA, and 2 mm PMSF) containing 1% digitonin for 1 h. The lysate was clarified by centrifugation at 10,000 × g for 10 min, and 100 μl of clarified lysate was sedimented on 5–30% w/v sucrose gradients under the same conditions as yeast. Fractions were analyzed by SDS-PAGE and immunoblotting with anti-HA antibody or anti-Sec61β antibody (described in a previous study (15 (link))).
Chemical Cross-linking Experiments—Yeast crude membrane fractions were resuspended in buffer containing 50 mm Hepes, pH 7.0, 150 mm KCl, and 1 mm PMSF. Ethylene glycobis(succinimidylsuccinate) (EGS, Pierce), was dissolved in anhydrous DMSO and diluted to the desired concentration. 1 μl of EGS was added into every 20 μl of protein-containing sample for 30 min at room temperature. The reactions were quenched for 15 min with 2 μl of 1 m Tris, pH 7.5. Samples were analyzed on a 4–20% SDS-PAGE and immunoblotted using standard procedures with mouse anti-His or rat anti-HA antibody conjugated to peroxidase (Sigma).
For mammalian cross-linking experiments, transfected COS-7 cells were grown in a 10-cm plate to ∼80% confluency and then lysed using a standard hypotonic lysis protocol. Briefly, cells were harvested in PBS, washed, incubated in hypotonic buffer (10 mm Hepes, pH 7.8, 10 mm potassium acetate, 1.5 mm magnesium acetate, 2 mm PMSF) for 10 min, and then passed through a 25-gauge syringe ten times. Nuclei and any remaining intact cells were separated from the lysate by centrifugation for 5 min at 3,000 × g, and the supernatant was then centrifuged for 10 min at 100,000 × g to pellet the membrane fraction. The membrane pellet was washed in HKM buffer (25 mm Hepes pH 7.8, 150 mm potassium acetate, 2.5 mm magnesium acetate, and 2 mm PMSF), repelleted at 100,000 × g, and resuspended to a final volume of 60 μl in HKM buffer. 10-μl membrane aliquots were used for each cross-linking reaction using the same conditions as above. Samples were analyzed on a 4–20% SDS-PAGE and immunoblotted using standard procedures with anti-HA antibody.

Rtn1p and Yop1p have slow diffusional mobility in the ER of yeast cells. A, typical FRAP of Sec63-GFP or Rtn1-GFP in S. cerevisiae cells expressed at endogenous levels. Images were taken before and then after the photobleach for the times indicated. The boxed region shows the area that was photobleached. B, fluorescence intensities normalized to prebleach values of FRAP analyses on yeast Sec63-GFP, Rtn1-GFP, and Yop1-GFP were plotted over time. Error bars indicate ± S.E.; n = 4 cells. C, fluorescence intensities normalized to prebleach values plotted over time of FRAP analyses on yeast Rtn1p in ATP-depleted (green) or non-depleted (orange) cells, compared with that of Sec63p-GFP (ATP depleted in blue; non-depleted in red). Error bars indicate ± S.E., n = 4 cells.
ATP Depletion Experiments—For yeast experiments, ATP was depleted by the addition of 10 mm 2-deoxy-d-glucose and 10 mm sodium azide (both from Sigma) for 2–5 min, and FRAP experiments were performed using the same parameters as described above. Similarly, for mammalian cell experiments, COS-7 cells were depleted of ATP as follows: transfected cells were washed twice in Opti-Mem serum-free media (Invitrogen) and then incubated with 50 mm 2-deoxy-d-glucose and 0.02% sodium azide in glucose-free imaging buffer (50 mm Hepes, pH 7.4, 150 mm potassium acetate, 2.5 mm magnesium acetate, and 1% fetal bovine serum). FRAP experiments were conducted in the same medium and completed within 5–30 min of treatment using the same parameters as above.