Abstract
Over the past few years, several reports have described the presence of F0F1 ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F1 sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary. Using different methods to prepare rat liver plasma membranes, which have been subjected to digitonin extraction, hr CN PAGE, immunoblotting, and mass spectrometry analysis, we demonstrate the presence of ecto-F0F1 complexes which have a similar molecular weight to the monomeric form of the mitochondrial complexes, containing both nuclear and mitochondrially-encoded subunits. This finding makes it unlikely that the enzyme assembles on the plasma membranes, but suggest it to be transported whole after being assembled in mitochondria by still unknown pathways. Moreover, the plasma membrane preparation enriched in basolateral proteins contains much higher amounts of complete and active F0F1 complexes, consistent with their specific function to modulate the HDL uptake on hepatocyte surface.
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Supplementary Material Fig. S1
Oligomycin sensitivity of F0F1 monomers and dimers from mitochondrial extracts. Mitochondrial proteins (500 μg) were extracted using 1 g/g of digitonin at 4 °C as described in Methods. Detergent extracts (5 μl) were separated by hr CN PAGE and gel slices were preincubated with or without oligomycin before in-gel activity staining as in (Wittig et al. 2007). (PPTX 139 kb)
Supplementary Material Table S1
List of the proteins identified by LC-MS/MS in the hr CN PAGE bands containing the F0F1 complexes extracted from mitochondria. The Table reports the Protein Score obtained in the Mascot search and the matched peptides for the proteins listed in Table 1 (main text). (XLS 36 kb)
Supplementary Material Table S2
List of the proteins identified by LC-MS/MS in the hr CN PAGE bands containing the F0F1 complexes extracted from plasma membranes. The Table reports the Protein Score obtained in the Mascot search and the matched peptides for the proteins listed in Table 2 (main text). (XLS 38 kb)
Supplementary Material Table S3
Proteins identified by LC-MS/MS in the hr CN PAGE bands containing the F0F1 complexes extracted from mitochondria. From the list, the proteins already reported in Table S1 are excluded. The Table reports for each protein hit the Protein Score obtained in the Mascot search, the matched peptides and the cellular localization of the identified proteins. (XLS 145 kb)
Supplementary Material Table S4
Proteins identified by LC-MS/MS in the hr CN PAGE bands containing the F0F1 complexes extracted from plasma membrane. From the list, the proteins already reported in Table S2 are excluded. The Table reports for each protein hit the Protein Score obtained in the Mascot search, the matched peptides and the cellular localization of the identified proteins. Proteins marked by (*) and (**) are found to be localized in plasma membrane rafts in references (Kim et al. 2006) and (Bae et al. 2004), respectively. (XLS 208 kb)
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Rai, A.K., Spolaore, B., Harris, D.A. et al. Ectopic F0F1 ATP synthase contains both nuclear and mitochondrially-encoded subunits. J Bioenerg Biomembr 45, 569–579 (2013). https://doi.org/10.1007/s10863-013-9522-z
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DOI: https://doi.org/10.1007/s10863-013-9522-z