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Fig. 4. Mfn1-containing complexes as analyzed by sucrose density gradient centrifugation and BN-PAGE. (A) Mitochondria prepared from Mfn1-FLAG or Mfn1-HA-expressing HeLa cells were solubilized with digitonin (w/o), mixed, and incubated with 0.4 mM GTP (GTP) for 1 hour at 30°C. The lysates were subjected to sucrose density gradient centrifugation in digitonin as described in Materials and Methods, and the separated fractions were analyzed by immunoblotting with anti-FLAG and anti-HA antibodies. Recovery of Mfn1 protein in the peak fractions was quantified and shown as % of total signals after centrifugation. (B) Mitochondria prepared from Mfn1-FLAG or Mfn1K88T-FLAG-expressing HeLa cells were solubilized by digitonin (w/o), and incubated with 0.4 mM GTP
S or GTP for 15 minutes or 1 hour at 30°C. In a separate experiment, after incubation with 0.4 mM GTP for 1 hour, 2 mM GTPS was added and incubated for an additional 1 hour at 30°C (GTP-GTPS). The reaction mixtures were subjected to sucrose density gradient centrifugation and analyzed as in A. (C) The mitochondria harboring Mfn1-FLAG were incubated with GTP at 30°C for the indicated time intervals. The reaction mixtures were then solubilized with digitonin and subjected to sucrose density gradient centrifugation, and analyzed as in A. (D) Mitochondria (mit) or salt-washed mitochondria (wash mit) were prepared from Mfn1-FLAG expressing HeLa cells, and incubated in the presence (+dig) or absence (–dig) of digitonin with (+GTP) or without (–GTP) GTP for 30 minutes at 30°C. The incubated mitochondria (for –dig) were solubilized by digitonin and subjected to sucrose density gradient centrifugation as in A. (E) Mitochondria (mit) or salt-washed mitochondria (wash mit) were prepared from HeLa cells expressing Mfn1-HA or Mfn1-FLAG, mixed, and incubated with GTP at 30°C for 1 hour (–dig). The membranes were solubilized by digitonin. Separately, mitochondria solubilized by digitonin (+dig) were also incubated with GTP. The reaction mixtures were subjected to sucrose density gradient centrifugation as above. Fractions 8 and 9 (peak 1), 11 and 12 (peak 2) or 14 and 15 (peak 3) were analyzed by immunoprecipitation using anti-HA antibodies, and subsequent immunoblotting using anti-FLAG antibodies as in Fig. 2. (F) BN-PAGE of mitochondria harboring Mfn1-FLAG or Mfn1-HA. Mfn1-harboring mitochondria were incubated with GTP as in D, and subjected to BN-PAGE. The separated protein complexes were analyzed by immunoblotting using anti-HA antibody. Marker proteins used were serum albumin (66 kDa), lactate dehydrogenase (140 kDa), catalase (232 kDa), apoferritin (440 kDa) and thyroglobulin (669 kDa).
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