Figure 1. ALIX-membrane interactions are LBPA- and calcium-dependent.

(A) Schematic representation of ALIX protein domains. (B) Recombinant ALIX_Bro1 and ALIX_V were incubated with liposomes (DOPC/DOPE/PI/LBPA; [5:2:1:2 mol]) for 2h at 4°C. Then, the liposome-bound protein was separated from free protein by floatation in sucrose gradients. Fractions were collected and analyzed by western blotting: (Lip) 50% of the fraction containing liposomes; (35%) 5.5% of the 35% sucrose cushion; (load) 12% of the fraction containing the load; (Std) 0.25 μg of the corresponding recombinant protein as standard. With LBPA present at the same level as in late endosomes (Kobayashi et al., 1998), 5% of total ALIX_Bro1 was found associated with liposomes after floatation, consistent with the characteristically weak and dynamic nature of protein-lipid interactions. (C–D) Liposome-binding of recombinant ALIX_Bro1 was analyzed as in (B), but in the presence of the indicated concentrations of free calcium (C), and quantified in (D). (E) A typical calcium titration curve monitored by ITC (upper panel) shows calcium-binding to ALIX_Bro1. The curve fit is illustrated in the lower panel, revealing that approximately 1 calcium atom was bound per ALIX_Bro1 molecule, and the thermodynamic values obtained from the curve fit are: n = 1.58 ± 0.06, K = 2.14*10⁶ ± 7.33*10⁵ M⁻¹, ΔH = 2892 ± 143.4cal/mol, ΔS = 39.2cal/mol/deg, K_D = 467 ±160nM. (F) Recombinant ALIX_Bro1 was incubated with liposomes containing LBPA as in (B) or not (DOPC/DOPE/PI; [7:2:1 mol]). Protein binding to liposomes was analyzed as in (B). (G) Data in (F) were quantified and are expressed as a percentage of LBPA values. (H) Binding of ALIX_Bro1 to liposomes containing 10mol% of the indicated phospholipids in 90mol% DOPC was analyzed as in (B). (I) Data in (H) were quantified and are expressed as in (G). All quantifications show means (±SEM) of 3 independent experiments (see also Fig S1).