Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments.
The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Andrés
A
Lorente-Rodríguez
Charles
C
Barlowe
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D022181:COP-Coated Vesicles / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; C487037:PLEKHA3 protein, human; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C037178:phosphatidylinositol 4-phosphate; C081864:SAC1 protein, S cerevisiae; D010744:Phosphoric Monoester Hydrolases