A Fluorescence-based Assay of Phospholipid Scramblase Activity

Summary

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Abstract

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin's scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin's scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

Introduction

The photoreceptor rhodopsin, a prototypical G protein-coupled receptor (reviewed for example in reference ¹), is the first phospholipid scramblase to be identified and biochemically verified ^2,3. Scramblases are phospholipid transporters that increase the intrinsically slow rate of transbilayer phospholipid movement to physiologically appropriate levels in a bidirectional, ATP-independent manner ^4-6. Examples of their actions can be found in the endoplasmic reticulum and bacterial cytoplasmic membrane where constitutive scrambling is needed for membrane homeostasis and growth, as well as for a variety of glycosylation pathways ⁵

Protocol

1. Preparation of Liposomes and Proteoliposomes

1. Liposome Formation
   1. Using a glass syringe, add 1,435 µl POPC (25 mg/ml, in chloroform) and 160 µl POPG (25 mg/ml, in chloroform) to a round bottom flask to obtain 52.5 µmol lipids in a molar ratio of POPC:POPG = 9:1.
   2. Dry the lipids for 30 min using a rotary evaporator at a rotation speed of 145 rpm (no water bath is needed for this volume of solvent), then transfer the flask to a vacuum desiccator for at least 3 hr, or overnight, at room temperature (RT).
   3. Hydrate the dried lipid film with 10 ml of 50 mM HEPES pH 7.4, 100 mM NaCl (henceforth referred to as buffer....

Results

We describe the reconstitution of opsin into LUVs to characterize its scramblase activity using a fluorescence-based assay. We analyze the results to place a lower limit on the rate of opsin-mediated phospholipid scrambling and to determine the oligomeric state in which opsin functionally reconstitutes into the vesicles.

To identify optimal reconstitution conditions, it is necessary to determine empirically the amount of deterge.......

Discussion

The scramblase activity assay enabled us originally to determine that opsin has phospholipid scramblase activity ². The assay also allowed us to characterize opsin's scramblase activity by testing specificity (we used a variety of NBD-labeled reporter lipids such as NBD-phosphatidylethanolamine, labeled with NBD on an acyl chain as shown for NBD-PC in Figure 1A, or on the headgroup, NBD-sphingomyelin or NBD- phosphatidylserine ²), the effect of vesicle lipid composition (e.g.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine	Avanti Polar Lipids	850457C	POPC
1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt)	Avanti Polar Lipids	840457C	POPG
1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine	Avanti Polar Lipids	810130C	NBD-PC
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid	VWR Scientific	EM-5330	HEPES
NaCl	Sigma	S7653-1KG	NaCl
Dodecyl-β-D-maltoside	Anatrace	D310 5 GM	DDM
Fluorimeter cuvettes	sigma	C0918-100EA	cuvettes
Spectrofluorometer	Photon Technology International, Inc.	fluorimeter
Sodium hydrosulfite technical grade, 85%	Sigma	157953-5G	dithionite
GraphPad Prism 5 software			Prism
Tris Base	VWR	JTX171-3	Tris
LIPEX 10 ml extruder	Northern Lipids, Inc.		Extruder
Whatman, Drain disc, PE, 25 mm	Sigma	28156-243	Disc support
Whatman Nuclepore Track-Etched Membranes, 0.4 µm, 25 mm diameter	Sigma	WHA110607	400 nm membrane
Whatman Nucleopore Track-Etched Membranes, 0.2 µm, 25 mm diameter	Sigma	WHA110606	200 nm membrane
sodium phosphate	Sigma	S3264-500G
VWR Culture Tubes, Disposable, Borosilicate Glass, 13 x 100 mm	VWR Scientific	47729-572	glass tubes
Perchloric acid	Sigma	30755-500ML
Ammonium Molybdate Tetrahydrate	Sigma	A-7302	ammonium molybdate
(+)-Sodium L-ascorbate	Sigma	A7631-25G	sodium ascorbate
Bio-Beads SM2 adsorbents	Bio Rad	1523920	polystyrene beads
2.0 ml Microtubes clear	VWR Scientific	10011-742	Reconstitution tubes
Reconstitution glass tube	VWR Scientific	53283-800	Reconstitution glass tubes
Zetasizer	Malvern		DLS