Freeze-fracture analysis of the effects of intermediates of the phosphatidylinositol cycle on fusion of rough endoplasmic reticulum membranes.

While searching for the identity of the effector of the putative GTP-binding protein involved in fusion of rough endoplasmic reticulum (RER) cell-free incubation conditions were found permitting fusion in a GTP-independent manner. Membrane fusion was obtained using medium required to study synthesis of phosphatidylinositol (PI). We now report on the effects of various co-factors and intermediates of the PI cycle on the interaction of rough microsomes. By freeze-fracture, fusion of rough microsomes was defined as the appearance of fracture-planes of membrane larger than those of unincubated membrane. Cytosine triphosphate (CTP, 3 mM) in the presence of 2 mM MnCl2 was most effective in stimulating fusion. Guanosine triphosphate (GTP) at the same concentration, could substitute for CTP to stimulate fusion, ATP, ITP, UTP and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) could not. When combined together in the same medium CTP potentiated the effect of GTP. Arachidonic acid (20 micrograms/ml) also stimulated fusion in the presence of MnCl2. This led to the appearance of large fracture-planes of membrane with a heterogeneous distribution of intramembranous particles. Other saturated fatty acids at the same concentration did not stimulate fusion. Phosphatidylinositol (PI, 50 micrograms) and 2 mM MnCl2 had a similar effect as arachidonic acid and MnCl2 in stimulating fusion. The PI effect was largely augmented in the presence of CTP. Our results are consistent with the concept that metabolism of phospholipids may modulate GTP-dependent fusion of RER membranes.