Membrane tubules of uniform diameter (60-80 nm) and various lengths (up to several micrometers) emanate from elements of the Golgi stack and trans Golgi network (TGN). These organelle membrane tubules are thought to be involved in membrane trafficking and maintenance of Golgi͞TGN architecture. The number of these tubules, and their frequency of formation, can be greatly enhanced by the fungal metabolite brefeldin A (BFA), an inhibitor of Golgi͞TGN-associated coated vesicle formation. We show here that BFA stimulation of Golgi and TGN membrane tubulation, and the resultant retrograde transport of resident Golgi enzymes to the endoplasmic reticulum, was potently inhibited by a number of membrane-permeant antagonists of phospholipase A 2 (PLA 2 ; EC 3.1.1.4) activity. In addition, PLA 2 inhibitors on their own caused a reversible fragmentation of the Golgi complex into juxtanuclear, stacked cisternal elements. We conclude from these observations that tubulation of Golgi complex and TGN membranes requires a PLA 2 activity, and that this activity may participate not only in Golgi tubule-mediated retrograde trafficking to the endoplasmic reticulum, but also in the maintenance of Golgi complex architecture.

We report here that a broad spectrum of phospholipase A 2 (PLA 2 ) antagonists produce a concentrationdependent, differential block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but have no acute affect on Tf uptake from the cell surface. At low concentrations of antagonists (ϳ1 M), Tf and TfR accumulated in centrally located recycling endosomes, whereas at higher concentrations (ϳ10 M), Tf-TfR accumulated in peripheral sorting endosomes. Several independent lines of evidence suggest that this inhibition of recycling may result from the inhibition of tubule formation. First, BFA-stimulated endosome tubule formation was similarly inhibited by PLA 2 antagonists. Second, endocytosed tracers were found in larger spherical endosomes in the presence of PLA 2 antagonists. And third, endosome tubule formation in a cell-free, cytosoldependent reconstitution system was equally sensitive PLA 2 antagonists. These results are consistent with the conclusion that endosome membrane tubules are formed by the action of a cytoplasmic PLA 2 and that PLA 2 -dependent tubules are involved in intracellular recycling of Tf and TfR. When taken together with previous studies on the Golgi complex, these results also indicate that an intracellular PLA 2 activity provides a novel molecular mechanism for inducing tubule formation from multiple organelles.

Previous studies have established a role for cytoplasmic phospholipase A 2 (PLA 2 ) activity in tubule-mediated retrograde trafficking between the Golgi complex and the endoplasmic reticulum (ER). However, little else is known about how membrane tubule formation is regulated. This study demonstrates that isotetrandrine (ITD), a biscoclaurine alkaloid known to inhibit PLA 2 enzyme activation by heterotrimeric G-proteins, effectively prevented brefeldin A (BFA)-induced tubule formation from the Golgi complex and retrograde trafficking to the ER. In addition, ITD inhibited BFA-stimulated tubule formation from the trans-Golgi network and endosomes. ITD inhibition of the BFA response was potent (IC 50 ϳ10 -20 M) and rapid (complete inhibition with a 10 -15-min preincubation). ITD also inhibited normal retrograde trafficking as revealed by the formation of nocodazole-induced Golgi mini-stacks at ER exit sites. Treatment of cells with ITD alone caused the normally interconnected Golgi ribbons to become fragmented and dilated, but cisternae were still stacked and located in a juxtanuclear position. These results suggest that a G-protein-binding PLA 2 enzyme plays a pivotal role in tubule mediated trafficking between the Golgi and the ER, the maintenance of the interconnected ribbons of Golgi stacks, and tubule formation from endosomes. INTRODUCTIONRecent studies have provided evidence that cytoplasmic Ca ϩ -independent phospholipase A 2 (PLA 2 ) enzymes play roles in tubule-mediated trafficking in the secretory and endocytic pathways . For example, a wide spectrum of PLA 2 antagonists, including those specific for cytoplasmic Ca ϩ -independent PLA 2 enzymes, are potent inhibitors of brefeldin A (BFA)-stimulated tubule formation from the Golgi complex, trans-Golgi network (TGN), and endosomes, and tubule-mediated trafficking pathways from these organelles (de Figueiredo et al., 1998(de Figueiredo et al., , 2000(de Figueiredo et al., , 2001Drecktrah and Brown, 1999;Polizotto et al., 1999). The mechanisms responsible for PLA 2 -mediated tubule formation likely include the localized accumulation of inverted coneshaped lysophospholipids (LPLs) that are generated by PLA 2 hydrolysis. In particular, LPLs on the cytoplasmic surfaces of organelle membranes could contribute to the generation of outward bending, thus initiating tubule formation (Burger, 2000;Huijbregts et al., 2000;Huttner and Schmidt, 2002;Brown et al., 2003). Support for this idea was provided by studies showing that inhibition of LPL reacylation by a Golgi-associated lysophospholipid acyltransferase (LPAT) also leads to increased tubule formation and retrograde trafficking (Drecktrah et al., 2003). These studies suggest that LPATs function to negatively influence membrane tubulation by limiting the accumulation of LPLs. Thus, PLA 2 and LPAT enzymes provide for direct positive and negative effects on membrane tubule formation, respectively.The exact identities of the Ca ϩ -independent PLA 2 and LPAT enzymes involved in tubule formation are unknown, and, moreover...