title: Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling.
abstract: Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.
authors.affiliation: From the ‡Laboratory of Respiratory Biology and.
authors.forename: Saiful M
authors.initials: SM
authors.lastname: Chowdhury
authors.affiliation: ¶Section on Molecular Medicine, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157.
authors.forename: Xuewei
authors.initials: X
authors.lastname: Zhu
authors.affiliation: From the ‡Laboratory of Respiratory Biology and.
authors.forename: Jim J
authors.initials: JJ
authors.lastname: Aloor
authors.affiliation: From the ‡Laboratory of Respiratory Biology and.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Azzam
authors.affiliation: From the ‡Laboratory of Respiratory Biology and.
authors.forename: Kristin A
authors.initials: KA
authors.lastname: Gabor
authors.affiliation: From the ‡Laboratory of Respiratory Biology and.
authors.forename: William
authors.initials: W
authors.lastname: Ge
authors.affiliation: From the ‡Laboratory of Respiratory Biology and.
authors.forename: Kezia A
authors.initials: KA
authors.lastname: Addo
authors.affiliation: §Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709;
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Tomer
authors.affiliation: ¶Section on Molecular Medicine, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157.
authors.forename: John S
authors.initials: JS
authors.lastname: Parks
authors.affiliation: From the ‡Laboratory of Respiratory Biology and fesslerm@niehs.nih.gov.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Fessler
mesh_terms: D064286:ATP Binding Cassette Transporter 1 / Q000172:deficiency* / Q000378:metabolism; D000818:Animals; D017353:Gene Deletion; D020868:Gene Silencing / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction / Q000187:drug effects; D051193:Toll-Like Receptors / Q000378:metabolism*
chemical_list: D064286:ATP Binding Cassette Transporter 1; D008024:Ligands; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009419:Nerve Tissue Proteins; D020543:Proteome; D051193:Toll-Like Receptors; C577833:Abca1 protein, mouse; C586438:stomatin-like protein 2, mouse