Scrambling of natural and fluorescently tagged phosphatidylinositol by reconstituted G protein–coupled receptor and TMEM16 scramblases

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 ([ 3 H]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 [ 3 H]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 [ 3 H]PI hydrolysis increased, indicating that [ 3 H]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 scram-bling of a chemically unmodified phospholipid by a reconstituted scramblase. D; PPR, protein to phospholipid ratio; TE, Triton extract, enriched in yeast ER membrane proteins.

CLPTM1L is a lipid scramblase involved in glycosylphosphatidylinositol biosynthesis

“…3F), indicating that CLPTM1L facilitates transbilayer movement of both lipids. Consistent with a previous study using nhTMEM16 and opsin (24), hydrolysis of GlcN-PI or PI by PI-PLC in CLPTM1L-containing proteoliposomes was a biphasic process (Fig. 3, E and F), with an initial rapid burst corresponding to hydrolysis of substrate in the outer leaflet followed by a slower phase in which molecules from the inner leaflet cross the membrane to the outer leaflet.…”

“…Hydrolysis of GlcN-PI and PI increased from ~50% to >80% with increased PPRs from 0-7 mg/mmol (Fig. 3G), similar to a previous study using nhTMEM16 and opsin (24). To address whether CLPTM1L can translocate other kinds of PLs, we tested nitrobenzoxadiazole (NBD)labeled PLs.…”

“…C16/C12-NBD-PI (NBD-PI) was synthesized from C16/C12-NBD-PC (NBD-PC) and myoinositol by phospholipase D (PLD)-mediated transphosphatidylation as previously described for synthesis of C16/C6-NBD-PI (24,50). An engineered microbial PLD carrying G186T/W187N/Y385R mutations, which can transfer the phosphatidyl group toward the 1-OH group of myo-inositol selectively to synthesize 1-PI, was used (51).…”

“…3A), we measured the scramblase activity of CLPTM1L in vitro. Bacterial PI-specific phospholipase C (PI-PLC) has been previously used as a membrane-impermeable topology probe for PI and GlcN-PI (2,22,23), and a scramblase assay using PI-PLC has been described to test activities of other scramblases, including opsin and nhTMEM16, towards [ 3 H]PI in reconstituted vesicles (24). In this assay system, since PI-PLC only accesses the outer leaflet of liposomes and because spontaneous translocation of [ 3 H]GlcN-PI across the membrane bilayer is expected to be negligible in a short time, about 50% of [ 3 H]GlcN-PI will be cleaved on adding PI-PLC.…”

Probing the Subcellular Distribution of Phosphatidylinositol Reveals a Surprising Lack at the Plasma Membrane

“…We attempted this approach with inositol lipids, using commercially available TopFluor®-fatty acid conjugated lipids. We reasoned this was a potentially viable approach, since native PI translocase and/or scramblase activities have been reported in mammalian cells, albeit with weaker activity compared to aminophospholipid translocases (Bütikofer et al, 1990; Wang et al, 2018).…”