Huntingtin associates with acidic phospholipids at the plasma membrane.

We have identified a domain in the N terminus of huntingtin that binds to membranes. A three-dimensional homology model of the structure of the binding domain predicts helical HEAT repeats, which emanate a positive electrostatic potential, consistent with a charge-based mechanism for membrane association. An amphipathic helix capable of inserting into pure lipid bilayers may serve to anchor huntingtin to the membrane. In cells, N-terminal huntingtin fragments targeted to regions of plasma membrane enriched in phosphatidylinositol 4,5-bisphosphate, receptor bound-transferrin, and endogenous huntingtin. N-terminal huntingtin fragments with an expanded polyglutamine tract aberrantly localized to intracellular regions instead of plasma membrane. Our data support a new model in which huntingtin directly binds membranes through electrostatic interactions with acidic phospholipids.

Actin-related protein (Arp2) inserts into artificial lipid membranes.

Arp2 is localized in the cytoplasm of eukaryotic cells where it controls actin dynamics. Computer analyses have suggested one possible lipid binding site, residues 185-202 of the primary amino acid sequence on Arp2, that could allow for membrane attachment/insertion. We expressed this region as a fusion protein with schistosomal glutathione S-transferase (GST) and investigated the interaction of this fragment with mixtures of dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylglycerol (DOPG) phospholipids in reconstituted lipid bilayers using differential scanning calorimetry (DSC). Calorimetric measurements showed that as the fusion protein increased, the main chain transition enthalpy decreased and the chain-melting temperature shifted, which is indicative of partial protein insertion into the hydrophobic region of the lipid membrane. This was confirmed using the Langmuir Blodgett technique (film balance) on lipid monolayers. The dissociation constant (K(d)) determined by the temperature jump method was approximately 1.1 microM.

Membrane fusion induced by the major lipid-binding domain of the cytoskeletal protein talin.

Secondary structure predictions have led to the identification of a major membrane-anchoring domain of the cytoskeletal protein talin spanning from amino acid 385 to 406. Using a synthetically derived peptide of this region, researchers have shown that it inserts into POPC/POPG phospholipid membranes with a partition coefficient of K(app)=1.1+/-0.2 x 10(5) M(-1) and has an average molar reaction enthalpy of DeltaH=-2.5 kcal/mol, as determined by monolayer expansion technique and isothermic titration calorimetry [J. Biol. Chem. 275, 17954]. We applied resonance energy transfer (RET) assays to analyze the fusogenic properties of this peptide by lipid mixing and used liposomes containing carboxyfluorescein to measure the contents leakage. We directly visualized talin peptide-induced vesicle membrane fusion using cryo-electron microscopy. This is the first example of a cytoskeletal protein domain that can trigger membrane fusion that might be of importance for understanding membrane targeting and motile events at the leading edge of the cell.