Figure 1.

Effect of cholesterol on hASCT2 reconstitution in proteoliposomes. (A) Purified WT hASCT2 was reconstituted in proteoliposomes prepared with the indicated amounts of CHEMS per mg phospholipids as described in Materials and Methods. Transport assay was started adding 50 μM of [³H]glutamine and 50 mM Na-Gluconate to proteoliposomes containing 10 mM glutamine. Transport was measured in 20 min, i.e., within the initial linear part of the time course. Transport rate was expressed as nmol/mg prot/min (∘, left y-axis). In the right y-axis, the internal volume of proteoliposomes used for the transport assay was calculated as described in Materials and Methods (•). (B) Experimental data obtained from experiments shown in (A) were analyzed subtracting each value corresponding to a specific CHEMS amount to the condition in which CHEMS was not present in the reconstitution mixture. Data were then plotted using non-linear Michaelis- Menten equation as described in Materials and Methods excluding data of high CHEMS amount, i.e., 100 and 150 μg/mg phospholipids, because out of the plot range. (C) Samples (a volume corresponding to 1% of the total reconstitution mixture) derived from (A) were subjected to SDS-PAGE and western blotting analysis, as described in Materials and Methods, for evaluating the incorporation of hASCT2 into proteoliposomes prepared with the indicated amount of CHEMS. Recombinant protein, harboring a 6His tag at the C-terminus, was detected using anti-His antibody. (D) Relative quantification of band intensity from western blotting of (C). In (A,B) data are means ± SD of three independent experiments. In (C), a representative image of three different experiments; in (D) data are means ± SD of three different western blotting analysis.