Experimental Procedures

Experimental Procedures

Analytical Fractionation – Livers from 3 male Balb/C mice were fractionated on ice as described {Croze, 1980 #407} with minor modifications. Minced livers (2-3 g) were homogenized into 6 ml of 0.5 M sucrose, 37.5 mM Tris-maleate, 1% dextran, 5 mM MgCl2 (homogenization buffer) with a Model 2000, variable speed polytron (OMNI International 2000, Winterbury, CT) at setting 4 for 40 sec. The homogenate was centrifuged at 5000 g for 15 min, giving rise to initial supernatant (S1) and pellet (P1). 6 ml of S1 was diluted 4-fold with homogenization buffer and centrifuged at 8500 g for 5 min, fractionating crude ER (S2) from contaminating mitochondria (P2). ER was purified by layering 24 ml of S2 on top of a discontinuous gradient consisting of 6 ml 2.0 M, 8 ml 1.5 M, and 8 ml 1.3 M sucrose in 37.5 mM Tris-maleate, 1% dextran, 5 mM MgCl2, pH 6.4, in a polyallomer centrifuge tube (25 x 89 mm, Beckman, …) and centrifugation for 120 min at 90000 g in a swinging bucket rotor (SW27, Beckman). ER-containing fractions were collected from both interfaces and pooled. The pooled fractions were again loaded on the same discontinuous sucrose gradient and centrifuged for a further purification step. For isolation of Golgi apparatus, the upper third of P1 was resuspended in a small volume of S1 by passage through a Pasteur pipet, 12x, layered over 2.7 ml of 1.2 M sucrose in 37.5 mM Tris-maleate, 1% dextran, 5 mM MgCl2, pH 6.4, in a polyallomer centrifuge tube (13 x 51 mm, Beckman) and centrifuged at 100000 g for 30 min in a swinging bucket rotor (SW60, Beckman). Golgi apparatus was collected from the interface. The lower half of P1 was resuspended in 1 mM Na2HCO3 with the polytron (setting 1), the volume adjusted to 10 ml with 1 mM Na2HCO3, and centrifuged at 4500 g for 10 min. The top third of the pellet was resuspended in 2-3 ml of the supernatant, mixed drop wise with 10 ml of a 81% solution of sucrose in H2O and transferred into a polyallomer centrifuge tube (25 x 89 mm, Beckman). On top of it was layered 3.5 ml each of 53.4, 48, 46, 44, 42.6 and 40% sucrose in H2O. The gradient was centrifuged for 90 min at 90000 g in a SW27 swinging bucket rotor. Plasma membrane was collected from the 40 - 42.5% interface, crude nuclei and mitochondria from the pellet. All fractionated organelles were washed twice in 20 ml of 10 mM Tris-HCl, pH 7.5. In preparation for 2D gel electrophoresis, aliquots of ER were pelleted in TLA 100 ultracentrifuge tubes (…x… mm, Beckman) at 100000 g for 30 min, supernatants were aspirated and the tubes stored at -80˚C. 10 µg of total protein from each fraction were separated by SDS-PAGE {Laemmli, 1970 #176} and transferred to nitrocellulose membranes. Western Blotting was performed as described {Dammann, 1996 #406} using the ECL system (Amersham) for detection. Primary antibodies were calnexin (?), Gp210 (kind gift of A. Wozniak, University of Alberta), mannosidase II (kind gift of T. Hobman, University of Alberta) rabbit polyclonal antisera at dilutions of 1:10000, 1:1000 and 1:2000, and porin (Calbiochem, San Diego, CA), HSP70 (Affinity BioReagents, Golden, CO) and Na+/K+-ATPase (?) monoclonal antibodies at dilutions of 1:1000 1:5000 and 1:200. Secondary antibodies were horseradish peroxidase conjugated goat anti rabbit and goat anti mouse (Jackson Immuno Research, West Grove, PA) at a dilution of 1:10000. For evaluation of the structural integrity and purity of the fractionated organelles, 50 µl aliquots were fixed for 2x 30 min in 2.5% glutaraldehyde, 1% OsO4 (both Polysciences, Warrington, PA) in 100 mM PIPES, pH 7.0, on ice in the dark. Samples were washed twice with 100 mM PIPES, pH 7.0, embedded in 0.5% low melting point agarose in 100 mM PIPES, pH 7.0, dehydrated and embedded in Spurr’s resin (…,…) for 24 hours at 70˚C. Ultrathin sections were contrasted with a 2% aqueous solution of uranyl acetate followed by 15 mM lead citrate and inspected in a … transmission electron microscope (Hitachi, Japan).

Two Dimensional Gel Electrophoresis and Image Analysis – All equipment for IEF (IPGphor, IPG strip holders and dry strips, pharmalytes) was from Amersham Pharmacia Biotech. (Uppsala, Sweden). Acrylamide and N, N’-bisacrylamide were from BioRad (Hemel Hempstead, UK), Tris, SDS, and glycine from Roche (Basel, Switzerland), Serdolit MB-1 from Serva (Heidelberg, Germany), all other chemicals from Sigma (St. Louis, MO). Aliquots of ER were solubilized for 3 hours at room temperature in 1 ml of 8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 15 mM DTT, 1 mM TCEP, 0.7% pharmalytes, trace of bromophenol blue (IEF buffer). IEF buffer was prepared freshly by deionizing 10 ml of the urea/thiourea/CHAPS solution with 0.2 g Serdolit MB-1 on a shaker for 10 min, and adding the other components after filtering and readjusting the volume. Insoluble material was removed by centrifugation at 40000 g for 45 min at 23˚C. The pellet was stored at -80˚C. The protein concentration of the supernatant was estimated with a … kit (BioRad, …). Semipreparative IEF was carried out under silicon oil after applying 300 µg of protein in a total volume of 400 µl per 18 cm strip. Strips with linear gradients of pH 3-10, 3.5-4.5, 4-5, 4.5-5.5, 5-6, 5.5-6.7, and 6-11 were used. All experiments were done in quadruplicate. After in gel rehydration for 14 hours at 30 V, IEF parameters were: 150 V, 1 hour; 500 V, 1 hour; 1000 V, 1 hour; gradient to 8000 V, 30 min; 8000 V to the steady state, depending on the pH interval used (48000 Vhrs for pH 3-10, 120000 Vhrs for narrow range strips, and 150000 Vhrs for pH 6-11 strips). IPG strips were equilibrated for 12.5 min in 10 ml of 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT, trace of bromophenol blue. Second dimension SDS-PAGE (11% T) was carried out simultaneously on 4 to 8 slab gels (230 x 180 x 1.5 mm) in a DALT tank (Hoefer, San Francisco, CA). Silver staining was performed as described {Blum, 1987 #24}. Gels were scanned with a … scanner (Amersham) at a resolution of 600 dpi, converted to 8 bit gray-scale mode and imported as TIF files into ImageMaster (version). The parameters for spot detection were: sensitivity, 9500; noise factor, 5; operator size, 95; background factor, 120. After spot detection, the images were edited manually (adding, removal, splitting of spots). Images from each series of gels were aligned, spots were excised manually in a laminar flow hood and matched spots from 2 to 4 gels were pooled prior to tryptic digestion.

Triton X-100 Partitioning of ER membrane proteins – to be done

In-gel enzymatic digestion – Bernd, Liang

Mass spectrometric analysis – Bernd, Liang

Confocal microscopy of…- to be done

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