Membrane interactions of the hydrophobic segment of diacylglycerol kinase epsilon

Diacylglycerol kinase epsilon (DGKε) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKε of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH₂. Flanking Lys residues mimic the natural setting of this peptide in DGKε, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic - but not zwitterionic - bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize - a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKε protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.     

The hydrophobic segment of Arabidopsis thaliana cluster I diacylglycerol kinases is sufficient to target the proteins to cell membranes

Diacylglycerol kinases (DGKs) catalyze the phosphorylation of diacylglycerol into phosphatidic acid. To fulfill their role in many signalling processes, DGKs must be located at, or in, membranes. Most mammalian DGKs are cytosolic and are recruited to membranes upon stimulation, except for epsilon type DGKs that are permanently membrane-associated through a hydrophobic segment. Nothing is known about the mechanism(s) involved in the membrane localization of plant DGKs. By fusion to fluorescent proteins, we show that two DGKs from cluster I in Arabidopsis thaliana possess amino-terminal hydrophobic segments that are sufficient to address them to endoplasmic reticulum membranes.     

Myristoylation of human LanC-like Protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin

Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-β-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.