Figure 1.
Schematic representation of flippase assay.
Flippase activity is measured by quenching fluorescently labeled PLs by sodium dithionite. (A) Energy independent activity -Reconstituted liposomes and proteoliposomes without flippase when treated with sodium dithionite, quenches half ∼50–55% the NBD-labeled PLs. In vesicles reconstituted with a functional flippase capable of bi-directional PL translocation there is a second phase of slow quenching of NBD labeled lipids to >55–100% (B) On addition of 0.1% (w/v) Triton X-100 to the symmetrically labeled liposomes (straight line) and proteoliposomes (dotted line) the vesicles are disrupted making all the PLs accessible to dithionite showing ∼100% quenching.
Figure 2.
Isolation of intact spinach chloroplast membrane.
(A) ATPase activity in isolated fractions. ATPase activity was measured as the amount of Pi released by ATP hydrolysis using Ames reagent which is measured at 820 nm. Mean values and the range from duplicate samples within one representative experiment are presented (B) SEM image of intact chloroplast (represented by arrows) which were fixed and coated with gold particles was observed under1000× magnification.
Figure 3.
Confirmation of vesicle intactness.
(A) Sucrose floatation gradient analysis, x-axis shows the interfaces between two sucrose step gradients and y-axis shows the corresponding percent total lipid and protein at each interface. (B) Collisional quenching of NBD fluorescence with iodide ions to determine the fraction of NBD-PC accessible on the outer leaflets of vesicles. Proteoliposomes (open squares) and liposomes (open circles) were reconstituted from Triton X-100-solubilized mixtures containing NBD-PC. Asymmetrically labeled liposomes (filled circles) were prepared by adding NBD-PC to preformed vesicles. The data are presented as modified Stern-Volmer plots, Fo is the fluorescence intensity of the sample in the absence of quencher, whereas ΔF is the fluorescence intensity at a given iodide ion concentration. The inverse of the y-intercept represents the fraction of NBD-PC that is accessible to the quencher. The assay was performed 2 independent times and p calculated was <0.01 (C&D) Liposomes and proteoliposomes were analyzed in 90Plus particle size analyzer. The DLS measurements showed liposomes (C) to be 110±10 nm and proteoliposomes (D) to be 150±12 nm.
Figure 4.
Localization of ATP independent and ATP dependent flippase activity in proteoliposomes reconstituted from TE of spinach chloroplast membranes.
(A) The ATP independent translocation of NBD-PC in intact chloroplast (trace a – Liposome, b – 30 µl TE). (P<0.0001) (B) No energy independent translocation of NBD-PG in proteoliposomes from intact chloroplast (trace b) and liposomes (trace a) (p<0.05) (C) Energy independent translocation of NBD-PC (trace c) and no energy independent translocation of NBD-PG (trace b) in proteoliposomes reconstituted with envelope membranes and liposomes (trace a) (p<0.001) (D) No energy independent translocation of either NBD-PC or NBD-PG in proteoliposomes reconstituted with thylakoids (p<0.01). (E) Proteoliposomes were treated with 20 mM dithionite to make inside labeled vesicles and NBD-PG flippase activity from in to out was monitored in an energy dependent manner. The bar diagram represents proteoliposomes reconstituted with envelope membrane (white) and thylakoids (black) quenched with dithionite (control), NBD-PG proteoliposomes incubated with 5 mM GTP, ADP, ATP and 10 mg/ml trypsin quenched with dithionite (p<0.05) (F) Inside labeled vesicles of envelope membrane and thylakoid reconstituted proteoliposomes were prepared and NBD-PC flippase activity from in to out was monitored in an ATP dependent manner. The bar diagram represents proteoliposomes reconstituted with envelope membrane (white) and thylakoids (black) quenched with dithionite (control), NBD-PC proteoliposomes incubated with 5 mM ATP and quenched with dithionite (p<0.05). All the experiments were carried out 2 independent times.
Figure 5.
Biogenic membrane flippase activity in intact chloroplasts.
(A) The level of translocation of NBD-PC increases with an increase in the amount of protein in the proteoliposome reconstituted with intact chloroplast (trace a – Liposome, b – 30 µl TE, c- 60 µl TE) (P<0.001) (B) Linear relationship between the amounts of TE to PPR in the reconstituted vesicles. (C) The extent of dithionite reduction of NBD-PC depends on protein to PL ratio (PPR). Flippase activity increases proportionately with an increase in PPR and saturates at a PPR∼400 mg/mmol. Above this value the percentage of fluorescence quenching remains unchanged for proteoliposomes. Kinetics of NBD-PC flipping - The calculation of half-life time was performed using the equation F(t) = F0−[A1 exp(−k1t)+A2 exp(−k2t)] where, F(t) is the fluorescence as a function of time and F0 is the fluorescence intensity at time = 0 s (i.e. initial fluorescence of the vesicles), k1 and k2 are the rate constants for the first (fast) and second (slow) phases respectively. A1 and A2 are the amplitudes of the fast and slow phases respectively. (D) The first phase half-life time remained constant at ∼0.6 min±0.05 min which represents the quenching of fluorescently labeled outer leaflet lipids. (E) The second phase half-life time decreased with increase in PPR suggesting that the kinetics of flipping increased and stabilized at a PPR of ∼400 mg/mmol. All the experiments were carried out 3 independent times.
Figure 6.
Effect of protease (trypsin) on flippase activity from spinach chloroplast membrane proteins.
(A) Trypsin treatment before reconstitution – TE was treated with 10 mg/ml trypsin and reconstituted (trace c), trace a and b represent liposome and untreated proteoliposome (control), (p<0.001) (B) Trypsin treatment after reconstitution – Proteoliposomes were incubated with trypsin and incubated at 37°C for 30 min and flippase assay was carried out, trace a and b represent liposome and untreated proteoliposomes, trace c shows proteoliposomes treated with trypsin, (p<0.01)(C) Bar diagram shows the decrease in flippase activity when proteoliposomes were treated with varying concentrations of trypsin, (p<0.01) (D) The kinetics of flipping was altered with increase in trypsin concentration.
Figure 7.
Effect of protein modifying reagents on the flippase activity.
Proteoliposomes and TE were incubated with desired concentration of protein modifying reagents at 37°C for 30 min. (A) Effect of DEPC on flippase activity (trace a- liposome, b- proteoliposome, c- proteoliposome treated with 20 mM DEPC) (B) Effect of AEBSF on flippase activity (trace a- liposome, b – proteoliposome, c- proteoliposome treated with 5 mM AEBSF) (C) Effect of PG on flippase activity ( trace a- liposome, b-proteoliposome, c-proteoliposome treated with 5 mM PG. (p<0.001) Effect of NEM on flippase activity. (D) Effect of NEM treatment on proteoliposomes (trace a- liposome, b- proteoliposomes, c- vesicle treated with 40 mM NEM). (E) Effect of dose dependent NEM treatment on proteoliposomes. (F) Effect of NEM treatment on TE extract prior to reconstitution. (trace a- liposomes, b- proteoliposomes, c- TE treated with 40 mM NEM) (p<0.01).
Figure 8.
Scramblase assay for inside labeled and outside labeled proteoliposomes.
(A) Inside labeled scramblase assay, both EGTA (control) treated (4 mM) and Ca2+ treated (2 mM) proteoliposomes showed same level of quenching. (B) Outside labeled scramblase assay, both showed same level of quenching confirming absence of scramblase activity (trace a – EGTA treated vesicles at t = 0 h, trace b - Ca2+ treated at t = 0, trace c- EGTA treated vesicles at t = 3 h, trace d – Ca2+ treated vesicles at t = 3 h).