Figure 3. Characterization of ALIX-membrane interactions.

(A) Liposome binding was analyzed as in Fig 1B for ALIX_Bro1, ALIX_Bro1I212D, ALIX_Bro1D97A, ALIX_Bro1D178A, ALIX_Bro1D314A and ALIX_Bro1D316A in the presence of 0., 0.1, 0.3 and 2μM free calcium. Binding is expressed as a percentage of the value obtained with 2μM free calcium. (B) ALIX_Bro1 was first pre-incubated with liposomes for 2h as in Fig 1B. The reaction mixture was then adjusted to 2M NaCl, 0.1M carbonate pH 11, 10mM EDTA or 2% NP40. ALIX_Bro1 remaining on the membrane was analyzed after floatation as in Fig 1B. (C) Quantification of membrane-bound ALIX_Bro1 in (B). Binding is expressed as a percentage of the control (Ctrl). (D) After binding to liposomes, membrane-bound and free ALIX_Bro1 were separated as in Fig 1B, and each was extracted with TX-114. Lanes 1–4: 0.25μg ALIX_Bro1 as standard; complete mixture before phase separation (input); water (Wat) and detergent (Det) phases after extraction. (E) Quantification of TX-114 extraction in (D), expressed as a percentage of the total protein in each condition. (F) Recombinant ALIX_Bro1 and ALIX_Bro1QQ were incubated in the presence or absence of 2.5μM calcium and liposomes lacking LBPA or containing 20mol% LBPA with or without 10% BrPC. Liposomes with membrane-bound protein were recovered by floatation and Trp fluorescence was measured. If indicated, 150 mM KI was added prior to the measurements. All quantifications show means (±SEM) of 3 independent experiments (see also Fig S3).