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Nordihydroguaiaretic acid (NDGA), a well known lipoxygenase inhibitor, actually has pleiotropic effects on cells, which include cell proliferation, apoptosis, differentiation, and chemotaxis. We and others have shown previously that this compound causes Golgi disassembly by an unknown mechanism. In this study, we show that, in parallel with Golgi disassembly, NDGA induces the accumulation of the microtubule minus-end-directed motor dynein-dynactin complex at the centrosome, where microtubules minus-ends lie. Concomitant with this accumulation, dynein-dynactin-interacting proteins, such as ZW10 and EB1, were also redistributed to the centrosomal region. In cells where microtubules were depolymerized by nocodazole, NDGA promoted the formation of filaments consisting of dynein-dynactin and its interacting proteins, suggesting that it stimulates the association of these proteins in an ordered, not random, manner. Loss of dynactin function abolished not only NDGA-induced redistribution in intact cells but also filament formation in nocodazole-treated cells. The latter finding implies that dynactin is a key molecule for the association between dynein-dynactin and its interacting proteins. In mitotic cells, NDGA induced robust accumulation of dyneindynactin and its interacting proteins at the spindle poles. These results taken together suggest that NDGA perturbs membrane traffic by affecting the function of the microtubule motor dynein-dynactin complex and its auxiliary proteins. To our knowledge, NDGA is the first case of a reagent that can modulate dynein-dynactin-related processes.

p125, a mammalian Sec23p-interacting protein, exhibits sequence homology with bovine testis phosphatidic acid-preferring phospholipase A1. In this study, we identified and characterized a new homologue of p125, KIAA0725p. KIAA0725p exhibited remarkable sequence similarity with p125 throughout the entire sequence determined but lacked an N-terminal proline-rich, Sec23p-interacting region. In vitrobinding analysis showed that KIAA0725p does not bind to Sec23p. KIAA0725p possessed phospholipase A1activity preferentially for phosphatidic acid. We examined the effects of overexpression of KIAA0725p on the morphology of organelles. Overexpression of KIAA0725p, like that of p125, caused dispersion of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus. Different from the case of p125, overexpression of KIAA0725p resulted in dispersion of tethering proteins located in the Golgi region and caused aggregation of the endoplasmic reticulum. Our results indicate that KIAA0725p is a new member of the phosphatidic acid-preferring phospholipase A1protein family and suggest that the cellular function of KIAA0725p is different from that of p125.

We examined the effects of short chain and long chain ceramides on the stability of the Golgi apparatus. Short chain ceramides, C2- and C6-ceramides, blocked brefeldin A-induced Golgi disassembly without affecting the rapid release of Golgi coat proteins, whereas they did not inhibit brefeldin A-induced tubulation of endosomes. Both short chain ceramides also retarded Golgi disassembly induced by nordihydroguaiaretic acid and nocodazole, suggesting that they stabilize the Golgi apparatus. In contrast to short chain ceramides, natural long chain ceramides, when incorporated into cells or formed within cells upon treatment with sphingomyelinase or metabolic inhibitors, enhanced brefeldin A-induced Golgi disassembly. These results suggest that sphingolipid metabolism is implicated in the stability of the Golgi apparatus.

N-Ethylmaleimide-sensitive factor (NSF) is an ATPase involved in many membrane fusion events within the exocytic and endocytotic pathways. In the present study we showed that NSF is associated with the nuclear envelope. Golgi-associated NSF was released from membranes upon incubation with Mg2+-ATP, reflecting the disassembly of a complex consisting of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). In contrast nuclear envelope-associated NSF in interphase cells was not released by the same treatment. During mitosis, however, it was released from nuclear membranes by Mg2+-ATP. These results suggest that the binding mode of nuclear membrane-associated NSF changes during the cell cycle.

The chemical cross-linking between the two cysteine residues at positions +290 and +302 of proOmpA was performed with N, N′-bis(3-maleimido-propionyl)-2-hydroxy-1,3-propanediamine. In the absence of the proton motive force (Δ \̃gmH +), the cross-linked proOmpA was only partially translocated into everted membrane vesicles, leading to accumulation of translocation intermediates. In the presence of Δ \̃gmH +, the cross-linked proOmpA was completely translocated. The translocated OmpA still possessed the cross-linked loop composed of 13 amino acid residues and the cross-linker. It is concluded that polypeptide chains need not be necessarily linear and fully expanded to be translocated.