Simon J P, Ivanov I E, Adesnik M, Sabatini D D
Department of Cell Biology, New York University School of Medicine, New York 10016, USA.
J Cell Biol. 1996 Oct;135(2):355-70. doi: 10.1083/jcb.135.2.355.
We have recently described a system that recreates in vitro the generation of post-Golgi vesicles from purified Golgi fractions obtained from virus-infected MDCK cells in which the vesicular stomatitis virus-G envelope glycoprotein had been allowed to accumulate in vivo in the TGN. Vesicle formation, monitored by the release of the viral glycoprotein, was shown to require the activation of a GTP-binding ADP ribosylation factor (ARF) protein that promotes the assembly of a vesicle coat in the TGN, and to be regulated by a Golgi-associated protein kinase C (PKC)-like activity. We have now been able to dissect the process of post-Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and another of vesicle scission, neither of which requires an ATP supply. The first stage can occur at 20 degrees C, and includes the GTP-dependent activation of the ARF protein, which can be effected by the nonhydrolyzable nucleotide analogue GTP gamma S, whereas the second stage is nucleotide independent and can only occur at a higher temperature of incubation. Cytosolic proteins are required for the vesicle scission step and they cannot be replaced by palmitoyl CoA, which is known to promote, by itself, scission of the coatomer-coated vesicles that mediate intra-Golgi transport. We have found that PKC inhibitors prevented vesicle generation, even when this was sustained by GTP gamma S and ATP levels reduced far below the K(m) of PKC. The inhibitors suppressed vesicle scission without preventing coat assembly, yet to exert their effect, they had to be added before coat assembly took place. This indicates that a target of the putative PKC is activated during the bud assembly stage of vesicle formation, but only acts during the phase of vesicle release. The behavior of the PKC target during vesicle formation resembles that of phospholipase D (PLD), a Golgi-associated enzyme that has been shown to be activated by PKC, even in the absence of the latter's phosphorylating activity. We therefore propose that during coat assembly, PKC activates a PLD that, during the incubation at 37 degrees C, promotes vesicle scission by remodeling the phospholipid bilayer and severing connections between the vesicles and the donor membrane.
我们最近描述了一种系统，该系统可在体外从感染病毒的MDCK细胞中获得的纯化高尔基体组分中重建高尔基体后囊泡的生成过程，在这些细胞中水泡性口炎病毒-G包膜糖蛋白已在体内高尔基体反面膜囊（TGN）中积累。通过病毒糖蛋白的释放来监测囊泡形成，结果表明其需要激活一种促进TGN中囊泡衣被组装的GTP结合ADP核糖基化因子（ARF）蛋白，并且受一种高尔基体相关的蛋白激酶C（PKC）样活性调控。我们现在已经能够将高尔基体后囊泡生成过程分解为两个连续阶段，一个是衣被组装和芽形成阶段，另一个是囊泡切割阶段，这两个阶段均不需要ATP供应。第一阶段可在20℃发生，包括ARF蛋白的GTP依赖性激活，这可由不可水解的核苷酸类似物GTPγS实现，而第二阶段不依赖核苷酸，且只能在较高的孵育温度下发生。囊泡切割步骤需要胞质蛋白，它们不能被棕榈酰辅酶A替代，已知棕榈酰辅酶A自身可促进介导高尔基体内部运输的衣被蛋白包被囊泡的切割。我们发现PKC抑制剂可阻止囊泡生成，即使在由GTPγS维持且ATP水平降低至远低于PKC的米氏常数（K(m)）的情况下也是如此。这些抑制剂抑制囊泡切割但不阻止衣被组装，然而要发挥其作用，它们必须在衣被组装发生之前添加。这表明假定的PKC的一个靶标在囊泡形成的芽组装阶段被激活，但仅在囊泡释放阶段起作用。PKC靶标在囊泡形成过程中的行为类似于磷脂酶D（PLD），一种已被证明即使在没有PKC磷酸化活性的情况下也能被PKC激活的高尔基体相关酶。因此我们提出，在衣被组装过程中，PKC激活一种PLD，在37℃孵育期间，该PLD通过重塑磷脂双层并切断囊泡与供体膜之间的连接来促进囊泡切割。
