TRANSBILAYER LIPID TRANSPORTERS

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DESCRIPTION (Adapted from applicant's abstract): The aminophospholipid phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane. Its exposure on the surface of blood cells promotes blood coagulation and provokes recognition and phagocytosis by macrophages as part of the normal processes of platelet activation and of programmed cell death, or apoptosis. PS is kept in the inner leaflet by the aminophospholipid translocase, an ATP-dependent enzyme which transports PS from the outer to the inner leaflet of the plasma membrane bilayer. The gene for this enzyme was recently cloned, revealing it to be a member of a previously unrecognized subfamily of P-type ATPases. The long-term goal of this project is to determine the biochemical and physiologic functions of this new subfamily of transporters. In yeast, there are five members of the subfamily, including a homolog of the mammalian aminophospholipid translocase. The genes coding for each of the other four subfamily members will be disrupted by homologous recombination to create mutant strains which will be analyzed for defects in morphology, physiology and biochemical function. Subfamily-specific consensus sequences will be used to generate specific primers for polymerase chain reactions to identify additional mammalian subfamily members. To establish whether there is any functional homology between related yeast and mammalian transporters, mammalian genes will be introduced into the corresponding yeast mutant to determine if they can compensate for the yeast defects. To gain information on the physiologic function of each mammalian subfamily member, the pattern of tissue expression and the chromosomal location of each gene will be determined. Antibodies will be prepared to the proteins encoded by subfamily member genes and used to analyze their pattern of expression in both yeast and mammalian cells. Finally, transgenic mice in which subfamily member genes have been disrupted will be generated to determine the physiologic consequence of ablation of the transporters in multicellular organisms.