Role of Lipid Modifications in Targeting Proteins to Detergent-resistant Membrane Rafts

TLDR

Detergent‑resistant membrane (DRM) rafts are sphingolipid‑ and cholesterol‑rich, Triton X‑100‑insoluble domains that likely exist as discrete liquid‑ordered microdomains in mammalian cells. The authors hypothesized that proteins with high affinity for ordered lipid environments would be targeted to rafts. Targeting to DRMs is driven by partitioning of saturated acyl chains, which adopt extended conformations compatible with liquid‑ordered domains, whereas branched prenyl groups are excluded from rafts. In MDCK cells, at least half of DRM proteins were palmitoylated, influenza hemagglutinin required all three palmitoyl sites for DRM association, while prenylated proteins such as Rap1, Rab5, Gβ, and Ras were excluded, demonstrating that multiple lipid modifications do not guarantee raft targeting.

Abstract

Sphingolipid and cholesterol-rich Triton X-100-insoluble membrane fragments (detergent-resistant membranes, DRMs) containing lipids in a state similar to the liquid-ordered phase can be isolated from mammalian cells, and probably exist as discrete domains or rafts in intact membranes. We postulated that proteins with a high affinity for such an ordered lipid environment might be targeted to rafts. Saturated acyl chains should prefer an extended conformation that would fit well in rafts. In contrast, prenyl groups, which are as hydrophobic as acyl chains but have a branched and bulky structure, should be excluded from rafts. Here, we showed that at least half of the proteins in Madin-Darby canine kidney cell DRMs (other than cytoskeletal contaminants) could be labeled with [3H]palmitate. Association of influenza hemagglutinin with DRMs required all three of its palmitoylated Cys residues. Prenylated proteins, detected by [3H]mevalonate labeling or by blotting for Rap1, Rab5, Gbeta, or Ras, were excluded from DRMs. Rab5 and H-Ras each contain more than one lipid group, showing that hydrophobicity alone does not target multiply lipid-modified proteins to DRMs. Partitioning of covalently linked saturated acyl chains into liquid-ordered phase domains is likely to be an important mechanism for targeting proteins to DRMs.