Monocyte lipid rafts contain proteins implicated in vesicular trafficking and phagosome formation

Abstract

Lipid rafts are membrane microdomains of unique lipid composition that segregate proteins with poorly understood consequences for membrane organization. Identification of raft associated proteins could therefore provide novel insight into raft-dependent functions. Monocytes process antigens for presentation to T cells by ingesting pathogens into calcium-dependent plasma membrane invaginations called "phagosomes" which develop by sequential fusion with the endoplasmic reticulum, early and late endosomes. We investigated the protein composition of Triton X-100 insoluble low density membranes of the monocyte cell-line THP-1 by matrix-assisted laser desorption/ionization-time of flight and tandem mass spectrometry. The ganglioside GM1 colocalized on the plasma membrane with the raft markers flotillin 1 and 2, which were enriched in low buoyant density fractions containing 52 identifiable proteins, 28 of which have not been reported in rafts, and nine of which are associated with the endoplasmic reticulum (ER). Remarkably, 27 of the 52 proteins are components of phagosomes, including the ER protein calnexin which we demonstrate is phosphorylated on serine 562, a switch controlling calcium homeostasis. The presence of the early and late endosome trafficking proteins Rab-1, and Rab-7 together with the late endosome protein LIMPII, indicate lipid rafts are present throughout endosome maturation. Identification of vacuolar ATP synthase, and synaptosomal-associated protein-23, proteins implicated in membrane fusion, together with the cytoskeletal proteins actin, alpha-actinin, and vimentin, and Rac 1, 2, and 3, regulators of cytoskeletal assembly, indicate monocyte lipid rafts contain the machinery to direct vesicular fusion and actin based vesicular migration throughout phagosome development.