Use of mass spectrometry-based lipidomics to probe PITP? (phosphatidylinositol transfer protein ?) function inside the nuclei of PITP?+/+ and PITP?-/- cells

Abstract

The mammalian phospholipid exchange protein PITPa (phosphatidylinositol transfer protein alpha), found in both extranuclear and endonuclear compartments, is thought in part to facilitate nuclear import of the PtdIns (phosphatidylinositol) consumed in the generation of proliferation-associated endonuclear diacylglycerol accumulations. Unlike phosphatidylcholine, endonuclear PtdIns is not synthesized in situ. However, despite progressive postnatal lethality of PITPa ablation in mice, PITPa-/- MEF (mouse embryonic fibroblasts) lack an obviously impaired proliferative capacity. We used ESI-MS (tandem electrospray ionization-MS) to monitor incorporation of the deuterated phospholipid precursors, choline-d9 and inositol-d6, into molecular species of whole cell and endonuclear phosphatidylcholine and PtdIns over 24 h to assess the contribution of PITPa to the nuclear import of PtdIns into MEF cells. In cells labelled for 1, 3, 6, 12 and 24 h fractional inositol-d6 incorporation into whole-cell PtdIns species was consistently higher in PITPa-/- MEF implying greater flux through its biosynthetic pathway. Moreover, endonuclear accumulation of PtdIns-d6 was apparent in the PITPa-/- cells and mirrored that in PITPa+/+ cells. Together, these results suggest that the essential endonuclear PtdIns import via PITPa can be accommodated by other mechanisms.

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Identifiers

ORCID for A.N. Hunt: orcid.org/0000-0001-5938-2152

ORCID for A.D. Postle: orcid.org/0000-0001-7361-0756

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