Snapp et al. 10.1073/pnas.0510657103. |
Supporting Figure 6
Supporting Materials and Methods
Supporting Figure 7
Fig. 6. Functional analysis of lectin-deficient Y108F mutant of Crt-GFP. (A) Crt knockout cells were transiently transfected with plasmids encoding ER-GFP, Crt-GFP, or Crt(Y108F)-GFP and analyzed 40 h later by pulse–chase labeling and native immunoprecipitation. Pulse-labeling (P) with [35S]methionine was for 30 min, followed by chase in unlabeled media for 90 min (C). Autoradiographs of total cell lysates (Upper) and the anti-Crt immunoprecipitates (Lower) are shown. The position of Crt-GFP is indicated. Coprecipitating proteins that transiently interact with Crt-GFP during the pulse and release from Crt during the chase period are indicated by asterisks. Note that these coprecipitating proteins are not seen with the lectin mutant Crt(Y108F)-GFP. Background bands (that were also seen in the ER-GFP-transfected cells) are indicated by the arrows. (B) An aliquot of total cell lysate from A was analyzed by immunoblotting using anti-Crt antibodies to confirm equal levels of steady-state expression for Crt-GFP and Crt(Y108F)-GFP.
Fig. 7. A comparison of the mobile fractions (Mf) of ER-GFP and Crt-GFP. The plot represents FRAP data sets for cells expressing the indicated protein and treated with puromycin for one hour. The Mf is the fraction of fluorescent proteins capable of diffusing into the photobleached region during the time course of the experiment. Thus, Mf is an operational value, and its primary utility is for comparing the effects of different conditions on the same protein. Data have been transformed to correct for the loss of total fluorescence (a bleach correction) and to scale the data such that the prebleach intensity is equal to 100% fluorescence intensity. The plot reveals that, during the time period of the experiment, the region of interest in the Crt-GFP-expressing cell recovers to 85% of prebleach fluorescence. In comparison, ER-GFP recovers to 95%.
Supporting Materials and Methods
Antibodies, Plasmids, and Drugs.
The construction of the GFP-tagged version of rat Crt (1) and mCFP-Sec61b (2) have been described previously. endoplasmic reticulum (ER)-GFP and ER-RFP contain in-frame fusions of the bovine prolactin signal sequence, mRFP or mGFP, and a KDEL ER retention sequence in the pCDNA3.1 vector. The Crt(Y109F)-GFP mutant was created by site-directed mutagenesis using the Quickchange kit from Stratagene as recommended by the manufacturer. Polyclonal rabbit anti-Crt (catalog No. PA3-900) was obtained from Affinity BioReagents, and monoclonal anti-GFP (JL-8) was obtained from Clontech. Horseradish peroxidase-conjugated anti-rabbit and mouse IgG antibodies were obtained from Amersham Pharmacia Biosciences. Pactamycin was a generous gift from E. Steinbrecher (Amersham Pharmacia, Peapack, NJ) and was used at 0.2 mM for at least 30 min. Puromycin (used at 1 mM) and cycloheximide (used at 0.5 mM) were obtained from Calbiochem, and cells were treated for at least 30 min. Castanospermine was obtained from Sigma, and cells were treated with 1 mM Castospermine and for at least 1 h. Cells were treated for no longer than 3 h for any of the drugs.Cells and Tissue Culture.
Wild-type Crt-expressing (K41) cells and Crt knockout cells (K42) (3, 4) were generously provided by the laboratory of Marek Michalak (University of Alberta, Edmonton, Canada). Cells were grown at 37°C in 5% CO2 in DMEM (Biofluids, Rockville, MD) supplemented with 10% FBS, glutamate, penicillin, and streptomycin. Stable clones of crt–/– cells expressing Crt-GFP were isolated by selection of transfected cells with Zeocin, after which they were sorted by FACS (using GFP fluorescence) to remove any nonexpressing cells. Transient transfections were performed by using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.Biochemical Analyses.
Total cell lysates for immunoblotting were prepared in 1% SDS, 0.1 M Tris, pH 8.0, by using cells in six-well plates at 80–90% confluence. Proteins were separated by using 12% Tris-tricine gels, transferred to nitrocellulose, probed with the indicated antibodies, and developed by using enhanced chemiluminescent reagents from Pierce. For pulse–chase analyses, cells in six-well plates were preincubated for 5 min in cysteine-, methionine-, and serum-free DMEM media (Gibco) before pulse-labeling with 400 uCi/ml (1 Ci = 37 GBq) [35S]methionine/cysteine (Translabel; ICN) for 15 min at 37°C. Cells were harvested immediately or washed into complete media containing serum and incubated for 60 min at 37°C. To harvest, cells were washed twice with 1× PBS and lysed with IP buffer (1% Triton X-100, 50 mM Hepes, pH 7.4, 100 mM NaCl) containing EDTA-free protease-inhibitor mixture (Roche). Lysates were clarified for 10 min at maximum speed in a microcentrifuge at 4°C and incubated for two hours at 4°C with polyclonal anti-Crt and protein A-Sepharose beads (Bio-Rad). The beads were washed four times in IP buffer, once in distilled water, eluted with SDS/PAGE sample buffer, and analyzed on 10% Tris/glycine minigels. In vitro transcription, translation, and translocation reactions using rabbit reticulocyte lysate and canine pancreatic rough microsomes (RM) were as described in ref. 5 and references therein. After translation for 1 h at 32°C, limited protease digestions for assessing Crt folding were in 0.5% Triton X-100 with 0.5 mg/ml proteinase K, as described in ref. 1.Live Cell Imaging and Photobleaching.
Cells were grown in eight-well Labtek chambers (Nunc) and imaged in phenol red-free RPMI medium 1640 supplemented with 10 mM Hepes and 10% FBS. Live cells were imaged on a temperature-controlled stage of a confocal microscope system (LSM 510; Zeiss) with either a ×40/1.3 NA oil objective or a ×63/1.4 NA oil objective. For imaging of cyan fluorescent protein (CFP) and GFP, a 413/488 dichroic mirror was used with low-intensity excitation (≈5 mW) of the 413-nm line of an Enterprise II ion laser (Coherent, Auburn, CA) with a 430–470 bandpass filter for CFP and the 488-nm line of a 40-mW Ar/Kr laser with a 500- to 550-nm bandpass filter for GFP. For simultaneous imaging of GFP and mRFP, the 543-nm line of a HeNe laser was used for mRFP excitation with a 488/543 dichroic mirror, and its emission was collected with a 560-nm long-pass filter. Alternatively, cells were imaged on a Leica SP2 TCS confocal microscope using a 63 × 1.4 NA oil objective and an objective heater. Cells were imaged with a 488-nm line of an Ar/Kr laser with a 500- to 550-nm bandpass emission for GFP. Qualitative fluorescence recovery after photobleaching (FRAP) experiments were performed by photobleaching a region of interest at full laser power of the 488-nm line and monitoring fluorescence recovery by scanning the whole cell at low laser power. No photobleaching of the cell or adjacent cells during fluorescence recovery was observed. Fluorescence recovery plots and diffusion (Deff) measurements were obtained by photobleaching a 4-mm-wide strip as described in refs. 6 and 7. Deff was determined by using an inhomogeneous diffusion simulation program described in ref. 7. To create the fluorescence recovery curves, the fluorescence intensities were transformed into a 0–100% scale in which the first postbleach time point equals 0% recovery, and the recovery plateau equals 100% recovery.To estimate Deff of Crt-GFP, we solved the Stokes–Einstein equation (D = (kT)/(6 πha), where D is the diffusion coefficient, k is Boltzmann’s constant, T is the temperature, h is the viscosity of the solution, and a is the hydrodynamic radius of the molecule for ER-GFP, with a = 2.3 nm (8). Assuming that temperature and viscosity remain constant, we estimated a of Crt-GFP, based on the separate a values for Crt (4.5 nm) (9) and GFP and assumed that, because GFP forms a distinct domain, a will be additive for the two hydrodynamic radii. From these inputs, the predicted Deff of Crt-GFP in the ER lumen in the absence of substrates was calculated.
Image analysis was performed by using nih image 1.63 and lsm image examiner software. Composite figures were prepared by using photoshop 7.0 and illustrator cs software (both from Adobe). Fluorescence recovery curves were plotted by using kaleidagraph 3.5 (Synergy Software).
Statistical Analyses.
To minimize the vagaries of cell-to-cell variables such as cell cycle stage or contact inhibition, we always selected flat, mononucleate, nonmitotic cells in cultures at between 40% and 70% confluency for analysis. Support for these selection criteria was the finding that distribution analysis (by Kolmorgov–Smirnov) on the sample set with the most accumulated data points showed an expected normal distribution calculated by using graphpad prism 4.0c software (MacKiev). Because such distribution analysis was not always feasible on the smaller data sets obtained for some of the different conditions, we have made the implicit assumption that a similar normal distribution is likely and, therefore, used a two-tailed Student t test (excel; Microsoft) to compare the different conditions. Variances of data sets were compared by using an F test (excel) to establish whether to use equal or nonequal variance t tests. Significance was tested by using a = 0.05.Mobile Fractions.
The mobile fraction (Mf) is the percent of total fluorescent protein capable of contributing to the fluorescence recovery of a photobleach region during the time course of a FRAP measurement. In this study, the Mf was calculated, as described in ref. 6, by comparing the fluorescence intensity in the photobleached region of interest before bleaching and after recovery (after correcting for any imaging-related bleaching of the sample). In this simplified calculation, the Mf can be <100% if an immobilized population exists or if sufficient time has not elapsed to get full recovery of fluorescence. Thus, the Mf clearly depends, at least partially, on the time course of the experiment and does not represent an absolute value, a point that needs to be kept in mind when interpreting this parameter.In our study, all cells examined exhibited mobile fractions >75% during the time course of the measurement (see Fig. 7). The cases where Mf fell <90% appear to simply reflect insufficient time for full recovery and not an actual immobilized population. This conclusion is supported by the fluorescence loss in photobleaching (FLIP) experiments (which occur over longer time frames than the FRAP experiments; Fig. 3A) and by inspection of a representative FRAP recovery plot (Fig. 7) that illustrates that recoveries are not entirely completed. These considerations suggest that the vast majority of the fluorescent protein in our measurements is mobile and that even the lowest values obtained (75–80%) are likely to be underestimates. A further complication in interpreting the Mf value is the geometrically complex structure of the ER. Photobleaching recovery is unlikely to be well modeled with simple diffusion on the mm scale. Hence, anomalous diffusion (10-13) may also explain, at least partially, the small changes in the Mf we have observed. Therefore, we have not interpreted the significance of any small changes in Mf, limiting our use of this parameter in this study solely to confirm that gross immobilization of fluorescent molecules did not occur under any of the conditions analyzed.
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