Stimulation of golgi membrane tubulation and retrograde trafficking to the ER by phospholipase A₂activating protein (PLAP) peptide

Abstract

Recent pharmacological studies using specific antagonists of phospholipase A(2) (PLA(2)) activity have suggested that the formation of Golgi membrane tubules, 60-80 nm in diameter and up to several microns long, both in vivo and in a cell-free cytosol-dependent reconstitution system, requires the activity of a cytoplasmic Ca(2+)-independent PLA(2). We confirm and extend these studies by demonstrating that the stimulators of PLA(2), melittin and PLA(2) activating protein peptide (PLAPp), enhance cytosol-dependent Golgi membrane tubulation. Starting with preparations of bovine brain cytosol (BBC), or a fraction of BBC that is highly enriched in tubulation activity, called the gel filtration (GF) fraction, that are at subsaturating concentrations for inducing tubulation in vitro, we found that increasing concentrations of melittin or PLAPp produced a linear and saturable stimulation of Golgi membrane tubulation. This stimulation was inhibited by cytosolic PLA(2) antagonists, including the Ca(2+)-independent PLA(2)-specific antagonist, bromoenol lactone. The stimulatory effect of PLAPp, and its inhibition by PLA(2) antagonists, was reproduced using a permeabilized cell system, which reconstitutes both cytosol-dependent Golgi membrane tubulation and retrograde trafficking to the endoplasmic reticulum (ER). Taken together, these results are consistent with the idea that cytosolic PLA(2) activity is involved in the formation of Golgi membrane tubules, which can serve as trafficking intermediates in Golgi-to-ER retrograde movement.

Role of Phospholipase A2 in Retrograde Transport of Ricin

“…In the toxicity assay (Figure 1), we could only use BEL at a concentration of up to 2.5 µM, as higher concentrations of BEL (5 and 10 µM) turned out to inhibit protein synthesis also in the absence of ricin. At a concentration of 2.5 µM, we could not observe any protective effects for BEL, although concentrations even lower than this (1 µM) has been shown to inhibit iPLA 2 activity [26,29,30,31]. In the sulfation assay, BEL could be used at a concentration of 10 µM without having any effect on total protein sulfation levels, and at this concentration BEL resulted in a significant increase in ricin sulfation.…”

“…Most of these studies have been performed using small molecule inhibitors of PLA 2 activity, such as ONO-RS-082 (ONO) and bromoenol lactone (BEL). However, the involvement of PLA 2 activity in these processes has been further demonstrated by the stimulation of Golgi membrane tubulation and retrograde trafficking to the ER by PLA 2 activating peptide (PLAP) [30] and by inhibition of LPAT (lysophosphatidylcholine acyltransferase), which catalyzes the reverse reaction of PLA 2 [31]. Furthermore, PLA 2 activity is induced by the addition of cholesterol, and is involved in the cholesterol-induced vesiculation of the Golgi apparatus [32].…”

Gβ1γ2 activates phospholipase A2-dependent Golgi membrane tubule formation

“…Either Gβ1γ2 does not stimulate membrane tubules or a cytosolic component (e.g., a PLA 2 ) is required to induce membrane tubule formation. Near background levels of membrane tubule formation are seen with low concentrations of BBC (sub-threshold levels), which can be used in combination with other factors that promote membrane tubule formation to achieve maximum Golgi membrane tubules (Polizotto et al, 1999). Therefore we tested whether Gβγ signaling requires a cytosol component by adding Gβγ to sub-threshold cytosol levels.…”

“…Grids were coded and counted blind. Golgi complexes on negative stain grids were identified by characteristic morphology (Banta et al, 1995; de Figueiredo et al, 1999; Polizotto et al, 1999), with a minimum diameter of 1 µm. Previous studies using immunogold labeling of Golgi-localized α-mannosidase II on whole mount preparations determined that reproducibly ~50% of the negatively stained profiles are intact Golgi complexes (de Figueiredo et al, 1999).…”

“…Indeed, a series of pharmacological studies using phospholipase A 2 (PLA 2 ) antagonists indicate that phospholipases are important for the formation of membrane tubules (de Figueiredo et al, 1998, 1999, 2000; Drecktrah and Brown, 1999; Polizotto et al, 1999). Recent studies have since identified specific phospholipase (PLA) enzymes that contribute to different levels of Golgi membrane tubule formation: cPLA 2 α (San Pietro et al, 2009), PLA2G6-A (Ben-Tekaya et al, 2010), and platelet activating factor acetylhydrolase Ib (PAFAH Ib) (Bechler et al, 2010).…”