Isolation of an Erythrocyte Membrane Protein that Mediates Ca2+-dependent Transbilayer Movement of Phospholipid

doi:10.1074/jbc.271.29.17205

Isolation of an Erythrocyte Membrane Protein that Mediates Ca²⁺-dependent Transbilayer Movement of Phospholipid*

From the Blood Research Institute of The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201

^‡To whom correspondence should be addressed:
Blood Research Institute, The Blood Center of Southeastern Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178.
Tel.: 414-937-3804; Fax: 414-937-6284.

Abstract

Elevation of intracellular Ca²⁺ in erythrocytes, platelets, and other cells initiates rapid redistribution of plasma membrane phospholipids (PL) between inner and outer leaflets, collapsing the normal asymmetric distribution. Consequently, phosphatidylserine and other lipids normally sequestered to the inner leaflet become exposed at the cell surface. This Ca²⁺-induced mobilization of phosphatidylserine to the surface of activated, injured, or apoptotic cells confers a procoagulant property to the plasma membrane, which promotes fibrin clotting and provides a signal for cell removal by the reticuloendothelial system. To identify the constituent of the membrane that mediates this Ca²⁺-dependent “PL scramblase” activity, we undertook purification and reconstitution of membrane component(s) with this activity from detergent extracts of erythrocyte ghosts depleted of cytoskeleton. Active fractions were identified by their capacity to mediate the Ca²⁺-dependent redistribution of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled PL between leaflets of reconstituted proteoliposomes. This PL scramblase activity co-eluted through multiple chromatographic steps with a single polypeptide of ∼37 kDa, which was purified to apparent homogeneity as resolved by silver staining. The activity associated with this protein band was inactivated by trypsin. The isolated protein reconstituted in proteoliposomes mediated nonselective, bidirectional transport of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-PL between membrane leaflets, with half-maximal activation between 20 and 60 μM Ca²⁺ (saturation >100 μM), mimicking the Ca²⁺-dependent transbilayer lipid movement intrinsic to the erythrocyte membrane.

Footnotes

↵* This work was supported in part by NHLBI Grant HL36946 from the National Institutes of Health (to P. J. S.) and an INSERM postdoctoral fellowship (to F. B.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PL

phospholipid

PC

phosphatidylcholine

PS

phosphatidylserine

PE

phosphatidylethanolamine

NBDPC

1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-snglycero-3-phosphocholine

NBD-PS

1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphoserine

OG

N-octyl-β-D-glucopyranoside

PIP₂

L-α-phosphatidylinositol 4,5-bisphosphate

IOVs

inside-out vesicles.
- Received March 22, 1996.
- Revision received May 6, 1996.