Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase

doi:10.1074/jbc.272.29.18240

Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase

A PROTEIN MEDIATING TRANSBILAYER MOVEMENT OF PLASMA MEMBRANE PHOSPHOLIPIDS*

From the Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201-2178

Abstract

The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca²⁺ is thought to play a key role in expression of platelet procoagulant activity and in clearance of injured or apoptotic cells. We recently reported isolation of a ∼37-kDa protein in erythrocyte membrane that mediates Ca²⁺-dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca²⁺ in the cytosol (Bassé, F., Stout, J. G., Sims, P. J., and Wiedmer, T. (1996) J. Biol. Chem.271, 17205–17210). Based on internal peptide sequence obtained from this protein, a 1,445-base pair cDNA was cloned from a K-562 cDNA library. The deduced “PL scramblase” protein is a proline-rich, type II plasma membrane protein with a single transmembrane segment near the C terminus. Antibody against the deduced C-terminal peptide was found to precipitate the ∼37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca²⁺-dependent PL scramblase activity was also demonstrated in recombinant protein expressed from plasmid containing the cDNA. Quantitative immunoblotting revealed an approximately 10-fold higher abundance of PL scramblase in platelet (∼10⁴ molecules/cell) than in erythrocyte (∼10³ molecules/cell), consistent with apparent increased PL scramblase activity of the platelet plasma membrane. PL scramblase mRNA was found in a variety of hematologic and nonhematologic cells and tissues, suggesting that this protein functions in all cells.

Footnotes

↵* This work was supported in part by Grant R01 HL36946 from the NHLBI, National Institutes of Health (to P. J. S. and T. W.) and a Grant-In-Aid from the American Heart Association (to T. W.).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.
↵‡ Recipient of Research Fellowship Award, American Heart Association, Wisconsin Affiliate.
↵§ To whom correspondence should be addressed: Blood Research Inst., Blood Center of Southeastern Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178. Tel.: 414-937-3850; Fax: 414-937-6284; E-mail:peter_s{at}smtpgate.bcsew.edu.
↵1 The abbreviations used are: PL, phospholipid(s); EST, expressed sequence tag; MBP, maltose binding protein; NBD-PC, 1-oleoyl-2-[6(7nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine; OG, N-octyl-β-d-glucopyranoside; PC, phosphatidylcholine; PS, phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid.
↵2 Q. Zhou, J. Zhao, J. G. Stout, P. J. Sims, and T. Wiedmer, unpublished data.
- Received April 25, 1997.
- Revision received May 19, 1997.