Lipid raft proteome reveals ATP synthase complex in the cell surface

Abstract

Since detergent-resistant lipid rafts are involved in pathogen invasion, cholesterol homeostasis, angiogenesis, neurodegenerative diseases and signal transduction, protein identification in the rafts could provide important information to study their function. Here, we analyzed detergent-resistant raft proteins isolated from rat liver by capillary liquid chromatography-tandem mass spectrometry. Out of 196 proteins identified, 32% belonged to the raft or plasma membrane, 24% to mitochondrial, 20% to microsomal, 7% to miscellaneous, and 17% are unknown proteins. For example, membrane-bound receptors, trimeric GTP-binding proteins, ATP-binding cassette transporters, and glycosylphosphatidylinositol-anchored proteins were identified in this analysis. Unexpectedly, there were many mitochondrial proteins, raising a new issue for the presence of mitochondrial rafts or the localization of mitochondrial proteins into plasma membrane rafts. We confirmed that ATP synthase alpha and beta were expressed on the surface of the plasma membrane in HepG2 hepatocytes by immunofluorescence, cell surface biotinylation, and cellular fractionation. They had two distinct biochemical properties, detergent insolubility and low density, suggesting that the ATP synthase complex might be located in plasma membrane rafts as well as in the mitochondria.

Amyloid precursor protein and amyloid β-peptide bind to ATP synthase and regulate its activity at the surface of neural cells

“…This result and the finding that a the faint band of ATP synthase subunit a was detected at the cell surface by immunostaining in the APPdeficient B103 cells (Figure 2e, lane 1) suggest the presence of an additional binding partner of ATP synthase subunit a at the neuronal cell surface. The punctate staining pattern of ATP synthase subunit a on neurons is in agreement with observations on the punctate appearance of cell surface ATP synthase subunit a on hepatocytes 21 and on a human umbilical vein endothelial cell line. 33 The localization of APP and ATP synthase subunit a at the cell surface of astrocytes was less punctate, and the continuous labeling showed more overlap with the staining of APP than in neurons ( Figure 5).…”

“…Thus, neural cells express ATP synthase subunit a not only in mitochondria, but also at the cell surface. The punctate appearance of ATP synthase subunit a immunostaining at the cell surface of primary hippocampal neurons is interesting with regard to previous observations that this enzyme is found at the cell surface of hepatocytes in lipid-rich microdomains 21 and in caveolae/lipid-enriched microdomains of non-neural cells. 53 Interestingly, APP has been reported to behave as an atypical lipid raft protein.…”

“…In addition, it was not only shown that ATP subunit a is present at the cell surface but also that the subunit a is a part of a competent multi-subunit F 1 F 0 -ATP synthase complex. [20][21][22]35 The transport of ATP synthase subunit a to the cell surface is sensitive to brefeldin A, a specific blocker of protein translocation from the endoplasmic reticulum to the Golgi apparatus. This observation indicates that cell surface-localized ATP synthase subunit a is transported via the secretory pathway to the cell surface, providing additional proof that the enzyme reaches the cell surface via a well-established processing pathway.…”

“…47 Our results showing IF 1 -sensitive extracellular generation of ATP by the glioma C6 cell line lead us to the conclusion that in this cell type there must also be a competent F 1 F 0 -ATP synthase complex at the cell surface, which could also be shown for other cell types. [20][21][22]35 Another known inhibitor of ATP synthase subunit a activity is angiostatin, a proteolytically derived 38 kDa fragment of plasminogen. 19,35 This interaction has been suggested to trigger the caspase-dependent apoptotic pathway in endothelial cells 48 and cytotoxicity in non-endothelial cells.…”

“…18 Although first described as a predominantly mitochondrial protein, the entire F 1 F 0 -ATP synthase complex has also been localized to the cell surface of endothelial cells, fibroblasts and hepatocytes, where the complex produces ATP extracellularly. [19][20][21][22] Our observations demonstrate a novel function for APP and Ab in regulating ATP synthase activity through interaction with the a subunit of ATP synthase. ATP is thus not only the main energy source of mammalian cells, but also an important extracellular signaling molecule with multiple functions.…”