Abstract
Members of the G protein-coupled receptor and TMEM16 (transmembrane protein 16) protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner. On reconstitution into large unilamellar vesicles, these proteins scramble more than 10,000 lipids/protein/s as measured with co-reconstituted fluorescent nitrobenzoxadiazole (NBD)-labeled phospholipids. Although NBD-labeled phospholipids are ubiquitously used as reporters of scramblase activity, it remains unclear whether the NBD modification influences the quantitative outcomes of the scramblase assay. We now report a refined biochemical approach for measuring the activity of scramblase proteins with radiolabeled natural phosphatidylinositol ([3H]PI) and exploiting the hydrolytic activity of bacterial PI-specific phospholipase C (PI-PLC) to detect the transbilayer movement of PI. PI-PLC rapidly hydrolyzed 50% of [3H]PI in large symmetric, unilamellar liposomes, corresponding to the lipid pool in the outer leaflet. On reconstitution of a crude preparation of yeast endoplasmic reticulum scramblase, purified bovine opsin, or purified Nectria haematococca TMEM16, the extent of [3H]PI hydrolysis increased, indicating that [3H]PI from the inner leaflet had been scrambled to the outer leaflet. Using transphosphatidylation, we synthesized acyl-NBD-PI and used it to compare our PI-PLC-based assay with conventional fluorescence-based methods. Our results revealed quantitative differences between the two assays that we attribute to the specific features of the assays themselves rather than to the nature of the phospholipid. In summary, we have developed an assay that measures scrambling of a chemically unmodified phospholipid by a reconstituted scramblase.
Highlights
Members of the G protein– coupled receptor and TMEM16 protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner
We exploited the ability of bacterial PI-specific phospholipase C (PI-PLC) to hydrolyze PI located in the outer leaflet of liposomes, whereas PI in the inner leaflet is protected from the action of the enzyme
The addition of PI-PLC to symmetric, large unilamellar liposomes containing a trace quantity of [3H]PI should result in hydrolysis of ϳ50% of [3H]PI, i.e. the portion of total PI that is present in the outer leaflet of the bilayer. [3H]PI in the inner leaflet is protected from PI-PLC action because the enzyme cannot cross the membrane bilayer, and the rate at which [3H]PI translocates spontaneously to the outer leaflet is too slow to be detected on the time scale of our experiments
Summary
Bacterial PI-PLC enzymes catalyze the cleavage of PI into diacylglycerol and inositol 1,2-cyclic phosphate [45, 46], which can be subsequently hydrolyzed to inositol 1-phosphate [45, 47]. Effect of the structure of the lipid reporter—Our results far indicate significant differences in the quantitative readout of scramblase activity obtained via the fluorescence-based versus PI-PLC– based assays Because both assays use topological probes that detect lipids only in the outer leaflet of the liposomes (dithionite does not enter vesicles containing opsin or nhTMEM16 as evinced by protection of trapped NBD-glucose [19, 24, 27], and PI-PLC is a large protein that cannot enter vesicles), they report on the scrambling of lipids from the inner to the outer leaflet, albeit with different characteristics. The results showed that ϳ80% of NBD-PC was reduced in both mock- and PI-PLC–treated proteoliposomes, demonstrating that the presence of the product of the PI-PLC reaction, diacylglycerol, had no effect on NBD-PC scrambling (Fig. 8B)
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