Function of the lipid transporter, PITP-beta in the early secretory pathway. (360G-Wellcome-079303_Z_06_Z)

Phosphatidylinositol transfer protein-beta (PITP-beta) is a transporter that can bind and transfer either phosphatidylinositol or phosphatidylcholine, thereby impacting upon lipid metabolism. Ablation of the gene encoding PITP-beta is embryonic-lethal indicating its essential function. We have identified PITP-beta localisation in early Golgi compartments, which suggests a role for this protein in the early secretory pathway where traffic is mediated by COP-1-coated vesicles. We want to identify potential binding partners of PITP-beta, using both affinity purification and yeast two-hybrid system, and characterise further any proteins purified. We will use siRNA to deplete PITP-beta levels and examine Golgi morphology and steady state dynamics of proteins of the COP1 pathway, in particular ARF GAP1, beta-COP, KDEL receptor and ARF1. The activity of ARF GAP1 is sensitive to membrane deformations which might be brought about by PITP-beta-mediated changes in lipid metabolism. Thus, perturba tion in lipid metabolism together with protein traffic will be analysed following depletion of PITP-beta levels. Our working hypothesis is that PITP-beta regulates local levels of diacylglycerol and/or phosphoinositides in the vesicular tubular compartments (VTC) and cis-Golgi, thereby regulating the COP-1 machinery. Using mutants deficient in binding and transfer of phosphatidylinositol, the specific function of PITP-beta in the secretory pathway will be investigated.