Muriel Marie-Paule, Dauphin Aurélien, Namekawa Michito, Gervais Annie, Brice Alexis, Ruberg Merle
INSERM, UMR S975, CRICM (formerly UMR S679), Paris, France.
J Neurochem. 2009 Sep;110(5):1607-16. doi: 10.1111/j.1471-4159.2009.06258.x. Epub 2009 Jul 1.
We examined the effects of wild-type and mutant atlastin-1 on vesicle transport in the endoplasmic reticulum (ER)-Golgi interface and vesicle budding from ER-derived microsomes using the temperature-sensitive reporter vesicular stomatitis virus glycoprotein (VSV-G), and the ability of purified atlastin-1 to form tubules or vesicles from protein-free phosphatidylserine liposomes. A GTPase domain mutation (T162P) altered the cellular distribution of the ER, but none of the mutations studied significantly affected transport from the ER to the Golgi apparatus. The mutations also had no significant effect on the incorporation of VSV-G into vesicles formed from ER microsomes. Atlastin-1, however, was also incorporated into microsome-derived vesicles, suggesting that it might be implicated in vesicle formation. Purified atlastin-1 transformed phosphatidylserine liposomes into branched tubules and polygonal networks of tubules and vesicles, an action inhibited by GDP and the synthetic dynamin inhibitor dynasore. The GTPase mutations T162P and R217C decreased but did not totally prevent this action; the C-terminal transmembrane domain mutation R495W was as active as the wild-type enzyme. Similar effects were observed in human embryonic kidney cells over-expressing mutant atlastin-1. We concluded that atlastin-1, like dynamin, might be implicated in membrane tubulation and vesiculation and participated in the formation as well as the function of the ER.
我们使用温度敏感的报告基因水泡性口炎病毒糖蛋白(VSV-G),研究了野生型和突变型atlastin-1对内质网(ER)-高尔基体界面处囊泡运输以及源自ER的微粒体出芽形成囊泡的影响,以及纯化的atlastin-1从无蛋白的磷脂酰丝氨酸脂质体形成小管或囊泡的能力。一个GTPase结构域突变(T162P)改变了ER的细胞分布,但所研究的突变均未显著影响从ER到高尔基体的运输。这些突变对VSV-G掺入由ER微粒体形成的囊泡中也没有显著影响。然而,atlastin-1也掺入了源自微粒体的囊泡中,这表明它可能与囊泡形成有关。纯化的atlastin-1将磷脂酰丝氨酸脂质体转化为分支小管以及小管和囊泡的多边形网络,这一作用受到GDP和合成的发动蛋白抑制剂dynasore的抑制。GTPase突变T162P和R217C降低了但并未完全阻止这一作用;C末端跨膜结构域突变R495W与野生型酶一样具有活性。在过表达突变型atlastin-1的人胚肾细胞中也观察到了类似的效应。我们得出结论,atlastin-1与发动蛋白一样,可能与膜管化和囊泡化有关,并参与了ER的形成以及功能。