Acute manipulation of Golgi phosphoinositides to assess their importance in cellular trafficking and signaling

摘要

Phosphoinositides are essential lipid regulators of trafficking and signaling pathways of all eukaryotic cells. Phosphatidylinositol 4-phosphate (Ptdlns4P) is an intermediate in the synthesis of several important phosphoinositide species but also serves as a regulatory molecule in its own right. Phosphatidylinositol 4-kinases are most abundant in the Golgi but are also found in the plasma membrane and in endocytic compartments. To investigate the role of Golgi Ptdlns4P in orchestrating trafficking events, we used a unique drug-inducible molecular approach to rapidly deplete Ptdlns4P from Golgi membranes by a recruitable Sad phosphatase enzyme. The utility of the system was shown by the rapid loss of Golgi localization of PH domains known to bind Ptdlns4P after Sac1 recruitment to the Golgi. Acute Ptdlns4P depletion prevented the exit of cargo from the Golgi destined to both the plasma membrane and the late endosomes and led to the loss of some but not all clathrin adaptors from the Golgi membrane. Rapid Ptdlns4P depletion in the Golgi also impaired but did not eliminate the replenishment of the plasma membrane Ptdlns(4,5)P_2 during phospho-lipase C activation revealing a hitherto unrecognized contribution of Golgi Ptdlns4P to this process. This unique approach will allow further studies on the role of phosphoinositides in endocytic compartments that have evaded detection using the conventional long-term manipulations of inositide kinase and phosphatase activities.