Polizotto R S, de Figueiredo P, Brown W J
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
J Cell Biochem. 1999 Sep 15;74(4):670-83.
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.
近期利用磷脂酶A2（PLA(2)）活性特异性拮抗剂开展的药理学研究表明，在体内以及无细胞的胞质溶胶依赖性重构系统中，直径为60 - 80纳米、长度可达数微米的高尔基体膜小管的形成需要一种胞质钙离子非依赖性PLA(2)的活性。我们通过证明PLA(2)的刺激剂蜂毒肽和PLA(2)激活蛋白肽（PLAPp）可增强胞质溶胶依赖性高尔基体膜成管作用，对这些研究进行了确认和拓展。以牛脑胞质溶胶（BBC）制剂，或BBC中高度富集成管活性的一部分（称为凝胶过滤（GF）组分）为起始材料，这些材料在体外诱导成管时处于亚饱和浓度，我们发现蜂毒肽或PLAPp浓度的增加会对高尔基体膜成管产生线性且饱和的刺激作用。这种刺激作用受到胞质PLA(2)拮抗剂的抑制，包括钙离子非依赖性PLA(2)特异性拮抗剂溴代烯醇内酯。使用通透细胞系统再现了PLAPp的刺激作用及其被PLA(2)拮抗剂抑制的情况，该系统重构了胞质溶胶依赖性高尔基体膜成管作用以及向内质网（ER）的逆行运输。综上所述，这些结果与胞质PLA(2)活性参与高尔基体膜小管形成的观点一致，高尔基体膜小管可作为高尔基体到内质网逆行运输的中间载体。
