Oxidation–reduction respiratory chains and ATP synthase complex are localized in detergent-resistant lipid rafts

Abstract

In order to detect and identify ubiquitous lipid raft marker proteins, we isolated lipid rafts from different mouse organs, including the liver, lung, large brain, and kidney, and analyzed their proteins via 2-DE. Many protein spots were determined to be ubiquitous in all of the lipid rafts, and were annotated via LC and MS/MS. Twelve proteins were identified as ubiquitous raft proteins, and most of these were determined to be mitochondrial proteins, including mortalin, prohibitin, voltage-dependent anion channel, ATP synthase, NADH dehydrogenase, and ubiquinol-cytochrome c reductase. Via immunoblotting, these proteins were shown to exist in detergent-resistant lipid rafts prepared using different organ tissues. Since these oxidationreduction respiratory chains and ATP synthase complex were detected in detergent-resistant lipid raft fractions which had been isolated from the plasma membrane but not from the mitochondria, and found in the cell surface when determined by immunofluoresence and immunohistochemistry, we conclude that plasma membrane lipid rafts might contain oxidation-reduction respiratory chains and ATP synthase complex.

Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery

“…Others have reported on cytosolic localization of GRP75 (also known as "mortalin"), where it may regulate the activity of p53 (15) and FGF (16). More recent proteomic analyses of detergent-resistant membrane fractions have suggested GRP75 is a ubiquitously expressed membrane lipid raft protein (17); however, the role of GRP75 in endocytic mechanisms remains unknown. MS data were confirmed by immunoblotting experiments, showing substantial enrichment of GRP75 in endocytic vesicles (Fig.…”

TRIM72 negatively regulates myogenesis via targeting insulin receptor substrate-1

“…Hind limb muscles (10)(11)(12) were surgically removed from 4-week-old female mice or 10-week-old male mice, washed three times in Dulbecco's PBS, chopped in 0.025% of collagenase type I (Sigma) with fine scissors, incubated at 371C for 90 min with stirring, and centrifuged at 300 Â g for 15 min. The pellet was resuspended in 10 ml of complete medium (Ham's F10 supplemented with 20% v/v FBS) and seeded into 100-mm gelatin-coated dishes.…”

“…2-DE, in-gel protein digestion, and LC-ESI-MS/MS were performed as previously described. 12 Adenovirus preparation and infection. An adenoviral vector with TRIM72 was constructed using ViraPower Adenoviral Expression System (Invitrogen, Carlsbad, CA, USA) under the control of cytomegalovirus (CMV).…”

“…Western blotting and immunofluorescence were performed as previously described. 12 For immunoprecipitation, cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 20 mM NaF, 10 mM Na 4 P 2 O 7 , 1 mM Na 3 VO 4 , 1% NP-40, 1 mM PMSF, and protease inhibitor cocktail. The whole-cell lysates (600 mg protein) were incubated with specific antibodies for 90 min and then with 50 ml of Protein A-sepharose or Protein G-agarose bead slurry for 90 min.…”

“…[6][7][8] To find novel lipid raft proteins, and gain new insights for the function of lipid rafts, we along with other groups have identified lipid raft proteins by proteomic analysis. [8][9][10][11][12] Previously, we identified a novel lipid raft protein, a receptor of globular C1q (gC1qR), by the differential proteomic analysis of lipid rafts from 3T3 L1 preadipocytes and adipocytes, and showed that gC1qR is an essential signaling molecule involved in adipogenesis and insulin signal transduction. 8 This result shows that the differential proteomic analyses of lipid rafts are very powerful tools to find novel signaling molecules involved in cellular differentiation, such as adipogenesis, neurogenesis, and myogenesis.…”