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Vol. 13, Issue 3, 739-754, March 2002

Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

James G. Alb Jr.,^* Scott E. Phillips,^* Kathleen Rostand, Xiaoxia Cui,^§ Jef Pinxteren, Laura Cotlin, Timothy Manning,^¶ Shuling Guo,^# John D. York,^# Harald Sontheimer,^¶ James F. Collawn, and Vytas A. Bankaitis^*@

 ^*Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7090;  Departments of Cell Biology,  ^§Biochemistry and Molecular Genetics, and  ^¶Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294;  Department of Physiology, University College London, London, United Kingdom; and  ^#Howard Hughes Medical Institute, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells. The best characterized mammalian PITPs are PITP and PITP, two highly homologous proteins that are encoded by distinct genes. Insights into PITP and PITP function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies. Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITP and PITP in murine cells. Contrary to expectations, we find that ablation of PITP function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism. Moreover, the data show that PITP does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor. These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane. Finally, the data demonstrate that PITP-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency. In contrast, we were unable to evict both PITP alleles from murine cells and show that PITP deficiency results in catastrophic failure early in murine embryonic development. We suggest that PITP is an essential housekeeping PITP in murine cells, whereas PITP plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence.

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These authors contributed equally to this work.

^@ Corresponding author. E-mail address: bktis{at}med.unc.edu.

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Molecular Biology of the Cell
Vol. 13, 739-754, March 2002
Copyright © 2002 by The American Society for Cell Biology

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