No abstract

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.

Abstract. Recent in vivo studies with the fungal metabolite, brefeldin A (BFA), have shown that in the absence of vesicle formation, membranes of the Golgi complex and the trans-Golgi network (TGN) are nevertheless able to extend long tubules which fuse with selected target organelles. We report here that the ability to form tubules (>7 #m long) could be reproduced in vitro by treatment of isolated, intact Golgi membranes with BFA under certain conditions. Surprisingly, an even more impressive degree of tubulation could be achieved by incubating Golgi stacks with an ATP-reduced cytosolic fraction, without any BFA at all. Similarly, tubulation of Golgi membranes in vivo occurred after treatment of cells with intermediate levels of NaN3 and 2-deoxyglucose. The formarion of tubules in vitro, either by BFA treatment or low-ATP cytosol, correlated precisely with a loss of the vesicle-associated coat protein ~/-COP from Golgi membranes. After removal of BFA or addition of ATE membrane tubules served as substrates for the rebinding of/3-COP and for the formation of vesicles in vitro. These results provide support for the idea that a reciprocal relationship exists between tubulation and vesiculation (Klausner, R. D., J. G. Donaldson, and J. Lippincott-Schwartz. 1992. J. Cell Biol. 116:1071-1080. Moreover, they show that tubulation is an inherent property of Golgi membranes, since it occurs without the aid of microtubules or BFA treatment. Finally the results indicate the presence of cytosolic factors, independent of vesicle-associated coat proteins, that mediate the budding/tubulation of Golgi membranes.

Abstract. Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energydependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for >12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.

Using a cell-free reconstitution system, we have characterized a cytosol- and ATP-dependent process that leads to the formation of membrane tubules from isolated Golgi complexes. These membrane tubules are uniform in diameter (50-70 nm) and morphologically identical to ones normally seen in cells and to those which are enhanced following brefeldin A treatment. Tubulation was strictly dependent on an activity present in an organelle-free extract of bovine brain cytosol and hydrolyzable ATP. Tubule formation was saturable with respect to both cytosol and ATP with half-maximal induction occurring at approximately 0.5 mg/mL cytosol and 10-20 microM ATP. Mild proteolytic treatment of Golgi membranes significantly reduced the extent of tubulation to subsequently added cytosol, suggesting that the tubulation activity interacts with Golgi-associated membrane proteins. The cytosolic tubulation activity was heat-labile, nondialyzable, and precipitated in ammonium sulfate. This activity could be followed through various chromatographic steps to yield fractions enriched in a major 40 kDa protein and several other minor proteins of approximately 80, 60, and 30 kDa. Monospecific antibodies against the 40K protein inhibited the cytosol-dependent tubulation of Golgi membranes in the cell-free system. Gel filtration chromatography suggests that the tubulation activity has a native molecular weight of approximately 125,000-140,000. These results establish the existence of cytosolic protein factors that regulate the formation of Golgi membrane tubules, and will provide the means for a biochemical dissection of membrane tubulation.