phosphatidylinositol-4-phosphate and 1-palmitoyl-2-oleoylphosphatidylcholine

Homologs of the phosphatidylinositol-4-phosphate-5-kinase (PIP5K), which controls a multitude of essential cellular functions, contain a 8 aa insert in a conserved region that is specific for the Saccharomycetaceae family of fungi. Using structures of human PIP4K proteins as templates, structural models were generated of the Saccharomyces cerevisiae and human PIP5K proteins. In the modeled S. cerevisiae PIP5K, the 8 aa insert forms a surface exposed loop, present on the same face of the protein as the activation loop of the kinase domain. Electrostatic potential analysis indicates that the residues from 8 aa conserved loop form a highly positively charged surface patch, which through electrostatic interaction with the anionic portions of phospholipid head groups, is expected to play a role in the membrane interaction of the yeast PIP5K. To unravel this prediction, molecular dynamics (MD) simulations were carried out to examine the binding interaction of PIP5K, either containing or lacking the conserved signature insert, with two different membrane lipid bilayers. The results from MD studies provide insights concerning the mechanistic of interaction of PIP5K with lipid bilayer, and support the contention that the identified 8 aa conserved insert in fungal PIP5K plays an important role in the binding of this protein with membrane surface. Proteins 2017; 85:1454-1467. © 2017 Wiley Periodicals, Inc.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amino Acid Sequence; Catalytic Domain; Conserved Sequence; Humans; Kinetics; Lipid Bilayers; Molecular Dynamics Simulation; Mutagenesis, Insertional; Phosphatidylcholines; Phosphatidylinositol Phosphates; Phosphotransferases (Alcohol Group Acceptor); Phylogeny; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Static Electricity; Structural Homology, Protein; Substrate Specificity; Thermodynamics

Lipids are unevenly distributed within eukaryotic cells, thus defining organelle identity. How non-vesicular transport mechanisms generate these lipid gradients between membranes remains a central question. Here using quantitative, real-time lipid transport assays, we demonstrate that Osh4p, a sterol/phosphatidylinositol-4-phosphate (PI(4)P) exchanger of the ORP/Osh family, transports sterol against its gradient between two membranes by dissipating the energy of a PI(4)P gradient. Sterol transport is sustained through the maintenance of this PI(4)P gradient by the PI(4)P-phosphatase Sac1p. Differences in lipid packing between membranes can stabilize sterol gradients generated by Osh4p and modulate its lipid exchange capacity. The ability of Osh4p to recognize sterol and PI(4)P via distinct modalities and the dynamics of its N-terminal lid govern its activity. We thus demonstrate that an intracellular lipid transfer protein actively functions to create a lipid gradient between membranes.

Topics: HeLa Cells; Humans; Lipid Metabolism; Liposomes; Lysine; Membrane Proteins; Oleic Acids; Phosphatidylcholines; Phosphatidylinositol Phosphates; Phosphatidylinositols; Receptors, Steroid; Saccharomyces cerevisiae Proteins; Sphingomyelins; Succinates

Phosphatidylinositol 4-phosphate (PtdIns(4)P) lipid is an essential component of eukaryotic membranes and a marker of the Golgi complex. Here, we developed metabolically stabilized (ms) analogs of PtdIns(4)P and the inositol 1,4-bisphosphate (IP(2)) head group derivative and demonstrated that these compounds can substitute the natural lipid fully retaining its physiological activities. The methylenephosphonate (MP) and phosphorothioate (PT) analogs of PtdIns(4)P and the aminohexyl (AH)-IP(2) probe are recognized by the PtdIns(4)P-specific PH domain of four phosphate adaptor protein 1 (FAPP1). Binding of FAPP1 to the PtdIns(4)P derivatives stimulates insertion of the PH domain into the lipid layers and induces tubulation of membranes. Both ms analogs and IP(2) probes could be invaluable for identifying protein effectors and characterizing PtdIns(4)P-dependent signaling cascades within the trans-Golgi network (TGN).

Topics: Adaptor Proteins, Signal Transducing; Binding Sites; Inositol Phosphates; Ligands; Lipid Bilayers; Magnetic Resonance Spectroscopy; Molecular Probes; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositol Phosphates; Phosphatidylinositols; Protein Conformation; Protein Structure, Tertiary; Recombinant Fusion Proteins; Signal Transduction