Lateral mobility of an amphipathic apolipoprotein, ApoC-III, bound to phosphatidylcholine bilayers with and without cholesterol

Abstract

The technique of fluorescence recovery after photobleaching was used to investigate the lateral mobility of a fluorescein-labeled amphipathic apolipoprotein, ApoC-II, bound to multibilayers prepared from dipalmitoyl phosphatidylcholine, egg phosphatidylcholine, and a 1:1 (molar ratio) mixture of egg phosphatidylcholine and cholesterol. In dipalmitoyl phosphatidylcholine bilayers the lateral diffusion coefficient (D) for the protein is about 2 X 10-9 cm2 sec1 at 200C and about 9 X 10-s cm2 sec1 at 450C. Plots The mobility of membrane components has drawn considerable attention in the recent literature (1, 2). Several reports on the lateral (3-7) and rotational diffusion (8, 9) of components in the plane of the membrane in model membrane systems have appeared. In model membranes, as opposed to biomembranes, it is possible in principle to examine simple membrane compositions-for example, one protein and one lipid membrane-and subsequently to increase the complexity of the system at will. In this manner, it is possible to learn about the influence of a given membrane component on the diffusion of other components in the membrane and also, by comparison to natural membrane diffusion results, to learn about the role that nonmembrane components in the cell play in the regulation of membrane component mobility.In the present communication we report the use of the technique of fluorescence recovery after photobleaching (FRAP) to study the lateral diffusion of an amphipathic apolipoprotein, ApoC-III, in bilayer membranes formed from dipalmitoyl phosphatidylcholine (Pam2PtdCho), egg-yolk phosphatidyl-choline (E-PtdCho), and an equimolar mixture of E-PtdCho and cholesterol (Chol). The results have been communicated in a preliminary form (10). FRAP has been used so far to study a wide range of natural membrane (11-21) and model membrane systems (3-7). It offers a convenient method for studying lateral diffusion and is particularly attractive from the points of view of experimental and theoretical simplicity (19,22).ApoC-III is an amphipathic peptide having a molecular weight of about 9000 and a known amino acid sequence (23,24). Its interaction with lipid membranes has been extensively studied (24). It interacts with E-PtdCho (25) MATERIALS AND METHODS ApoC-III was isolated according to the procedure of Brown et al. (28) and was labeled with fluorescein by reaction of the protein (1 mg/ml) in 0.1 M sodium borate buffer at pH 9.0 with a 100-fold molar excess of fluorescein isothiocyanate (Baltimore Biological Laboratory Division, Becton-Dickinson) for 1 hr at 250C. After reaction, the labeled protein (dye/protein ratio = 2) was separated from unreacted dye by filtration through a column of Sephadex G-25 using Dulbecco's Ca2+-and Mg2+-free phosphate-buffered saline.The phospholipids used in this study were isolated or synthesized by Tom Isac in the laboratory of D. Papahadjopoulos. Pam2PtdCho was synthesized as described by Robles and Van Den Berg (29) and E-PtdCho was isolated from egg yolks as described by Papah...

Cell surface properties of HLA antigens on Epstein-Barr virus-transformed cell lines.

“…sured values of D are in the range 10-9 to 10-10 cm2/sec, consistent with previous results in the literature for HLA antigens (51) and with values commonly reported for mobile membrane proteins (52). It is likely that the 10-9 cm2/sec value for D is that for a protein unhindered by cytoskeletal components because this is comparable to the values obtained for proteins in cholesterol-containing model membranes (53,54). The smaller values probably reflect some hindrance of the motion of the protein.…”

H-2Kk and vesicular stomatitis virus G proteins are not extensively associated in reconstituted membranes recognized by T cells.

“…The rate oflateral diffusion of these proteins is comparable to rates observed for other membrane proteins in similar lipid mixtures (27)(28) patched with unlabeled 114.1 antibody (first-step) and rhodamine-conjugated F(ab')2 rabbit anti-mouse antibody (Cappel, Cochranville, PA) (second-step, abbreviated RFRAM henceforth); (3,4) unlabeled G patched with rabbit anti-G and rhodamine-conjugated F(ab')2 goat anti-rabbit (Cappel) (abbreviated RFGAR henceforth); (5,6) F1M H-2Kk; (7,8) FITC-G; (9,10) FITC-H-2Kk patched with alloantiserum against H-2Kk and RFRAM (fluorescein fluorescence); (11,12) FITC-G patched with rabbit anti-G and RFGAR (fluorescein fluorescence); (13,14) FITC-H-2Kk and unlabeled G; (15,16) FITC-G and unlabeled H-2Kk; (17,18) FITC-H-2Kk and unlabeled G (fluorescein fluorescence) with the G protein patched with rabbit anti-G and RFGAR; (19,20) A strong specific interaction between viral proteins and H2Kk molecules in a reconstituted membrane would provide strong support for any theory that required close proximity of these two molecules in the target membrane during specific elicitation of cytotoxic T cells. On the other hand, freely and independently diffusing viral proteins and H-2Kk molecules are also consistent with a possible close proximity of these molecules as a prerequisite for specific elicitation of CTL (32).…”

The nucleolar detention pathway

“…Early work focused on the analysis of cell surface particles and utilized fluorescent dyes to monitor mobility during recovery after photobleaching. [1][2][3][4][5] Advances in time-lapse imaging technology and the cloning of green fluorescent protein 6 has allowed scientists to pass (Fig. 1A).…”