Digitonin

Cells grown in 6-well plates were washed with PBS and metabolically labeled (Easytag Express [35S]-Met/Cys protein labeling, Perkin Elmer, Waltham, MA) with 100 Ci/ml for 20 min. Cells were lysed in digitonin lysis buffer (140 mM NaCl, 20 mM Tris [pH 7.6], 5 mM MgCl₂, and 1% digitonin (Merck Millipore)) and cleared from membrane debris at 13,000 rpm for 30 min at 4 °C. Lysates were incubated with anti-His tag antibody for 2 h at 4 °C in an overhead tumbler before immune complexes were retrieved by protein G-sepharose (GE Healthcare, Chicago, IL). Sepharose pellets were washed four times with increasing NaCl concentrations (0.15–0.5 M in lysis buffer containing 0.2% detergent). EndoH (New England Biolabs) treatment was performed as recommended by the manufacturer. Prior to loading onto a SDS-PAGE immune complexes were dissociated at 95 °C for 5 min in a DTT (40 mM) containing sample buffer. Fixed and dried gels were exposed overnight to a phosphor screen, scanned by Typhoon FLA 7000 (GE Healthcare). For better visualization of the results, contrast and brightness were adjusted. A long exposure to X-ray film was used for autoradiography. Images were quantified using the ImageJ software^{83 (link)}.

The isolated thylakoid membranes were fractionated into grana and stroma fractions with digitonin. The thylakoid samples were diluted in BTH buffer [25 mM Bis/Tris/HCl (pH 7.0), 20% (w/v) glycerol, 0.25 mg/ml Pefabloc and 10 mM NaF] to a concentration of 1 mg/ml. An equal volume of 2% digitonin (Merck Life Sciences, Keilaranta 6, Espoo, Finland) in BTH buffer was added to the sample to achieve the final concentration of 0.5 mg/ml of Chl and 1% digitonin in the sample. The sample was solubilized for 8 min at room temperature (RT) in continuous mixing. The insoluble (grana) and soluble (stroma thylakoids) fractions were separated by centrifugation at 18,000×g for 20 min, at 4°C. After centrifugation, 10 µl of supernatant was added to 990 µl of buffered acetone (80% acetone and 2.5 mM HEPES/KOH pH 7.6), and the Chl concentration and Chl a/b ratio were determined by measuring the absorbance at wavelengths of 663.6, 646.6 and 750 nm. Chl a and b concentrations were calculated using the following formulas—Chl a: (1/10)*[12.25*(A664-A750) − 2.55*(A646-A750)]; Chl b: (1/10)*[20.31*(A646-A750) − 4.91*(A664-A750)] according to Porra et al. (1989) .

S10-3 cells were seeded onto glass coverslips and transfected 24 h later with ORF3 expression vectors or co-transfected with pUHD15-1 [51 (link)] and pUHD-Cp7 plasmids allowing the expression of HCV core-p7 region, as described previously [30 (link)]. Forty-eight h post-transfection cells were fixed with 2% paraformaldehyde (Sigma-Aldrich) for 10 min and then permeabilized, totally, with either 0.5% saponin or 0.2% digitonin (Sigma-Aldrich) or selectively with 0.01% digitonin. Cells were then washed and incubated 15 min at 20°C in blocking buffer containing 3% bovine serum albumin in PBS. Indirect immunofluorescence was then performed by one-hour incubation at 20°C with primary antibody, followed by 3 PBS washes and incubation with the secondary antibody as described above.

Stable KD cell pellets were incubated in digitonin containing buffer (150 mM NaCl, 50 mM Hepes, and 50 μg/mL digitonin (Sigma)) end-over-end for 10 min at 4 °C. Then, homogenates were centrifuged at 980 × g for 4 min at 4 °C. Supernatants were again centrifuged at 17,000 × g for 10 min at 4 °C and to allow removal of any remaining cellular debris. Supernatants were kept as cytosolic fractions and used for subsequent genomic and mitochondrial DNA analysis. Purity of the fractions was validated through western blotting of a lysosomal marker (LAMP1), a mitochondrial marker (TIMM23), and a cytosolic marker (Tubulin).

Cells were washed by PBS, scraped and centrifuged at 600×g for 5 min. Cell pellets equal to 500 µg of total protein were suspended in 70 µl lysis buffer (50 mM NaCl, 50 mM imidazole, 2 mM ɛ-aminocaproic acid, 1 mM EDTA, pH 7.0). Protein complexes were solubilized by digitonin (Sigma-Aldrich) at the final concentration of 6 g digitonin/g total protein. Samples were incubated at 4 °C for 20 min and cell debris removed by centrifugation at 30,000×g for 30 min at 4 °C. Samples were supplemented by glycerol (10%), ɛ-aminocaproic acid (50 mM) and Coomassie Brilliant Blue G-250 (0.5%; Serva, Heidelberg, Germany). Protein complexes (20 µg of total cell proteins) were separated by BN-PAGE in 4–13% polyacrylamide gradient gels as described earlier^{74 (link)} using a Mini-PROTEAN III apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Immunodetection procedure and the list of used antibodies was virtually identical to that listed above and published previously^{75 (link),76 (link)}. The modification included the use of different antibodies: anti-NDUFB8 Ab and anti-UQCRC2 Ab were used for the detection of complex I and III, respectively.

Wild-type S2 cells (1.5×10⁶) were plated on coverslips and permeabilized with 10 µg/ml digitonin (D141, Sigma-Aldrich) in KRB for 2 h at 26°C. Subsequently, cells were fixed and Sec bodies were visualized by immunostaining of Sec16. To test the effect of ATP, AMP or adenosine on Sec body formation, the semi-intact cells were incubated in the presence of 0.5 mM ATP (A1852, Sigma-Aldrich), 0.5 mM AMP (01930, Sigma- Aldrich) or 0.5 mM adenosine (A9251, Sigma-Aldrich). Note that the digitonin was not removed. The permeabilization efficiency was determined by using the non-membrane-permeable dye TO-PRO-3 iodide (T3605, Thermo Fisher Scientific) (Fig. 6A). The import buffer used in the SIC system was 20 mM HEPES, 110 mM KAc, 2 mM MgAc, 5 mM NaAc and 0.5 mM EGTA (pH was set at either 6.0 or 7.4 with KOH). For the RNA degradation experiment in the SIC system, we incubated S2 cells on coverslips for 2 h at 26°C in Schneider's medium containing 10 µg/ml digitonin (Sigma-Aldrich) with or without 0.25 U/ul RNase 1 (EN0602, Thermo Fisher Scientific).