Inhibition of Membrane Tubule Formation and Trafficking by Isotetrandrine, an Antagonist of G-protein-regulated Phospholipase A₂ Enzymes

Abstract

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...

Meltrin β (ADAM19) mediates ectodomain shedding of Neuregulin β1 in the Golgi apparatus: fluorescence correlation spectroscopic observation of the dynamics of ectodomain shedding in living cells

“…1992). ITD inhibits BFA‐induced tubule formation from the Golgi complex and retrograde trafficking to the ER and also inhibits BFA‐stimulated tubule formation from the Golgi apparatus to the ER (Chan et al . 2004).…”

The Role of GRASP55 in Golgi Fragmentation and Entry of Cells into Mitosis

“…Inhibiting phospholipase A2, and blocking protein transport from ER to the Golgi also disconnect Golgi stacks, but it is not known whether these processes are required for mitotic entry (Chan et al, 2004;Marra et al, 2007). Stacks of Golgi cisternae in Drosophila S2 cells that are unlinked (dispersed) undergo severing by an actin-dependent process, and surprisingly, this event is necessary for entry into mitosis .…”

Gβ1γ2 activates phospholipase A2-dependent Golgi membrane tubule formation

“…Previous studies have shown that ITD inhibits BFA-stimulated Golgi membrane tubules in mammalian cells (Chan et al, 2004). To determine if ITD similarly inhibits cytosol-stimulated Golgi tubules in vitro , we tested a range of ITD concentrations with varying BBC concentrations.…”

“…ITD has been used to inhibit inflammatory signaling, by decreasing Gβγ activation of PLA 2 enzymes (Hashizume et al, 1991). More recent studies show ITD inhibition of BFA-stimulated Golgi membrane tubule formation, suggesting a broader role of Gβγ regulation of PLA 2 enzymes (Chan et al, 2004). Here we further explore the hypothesis that ITD inhibits Gβγ activation of PLA 2 enzymes involved in the formation of Golgi membrane tubules.…”