Novel physiological roles of acyl-CoA : lysophosphatidic acid acyItransferases.

高尔基复合体的结构完全取决于它与其他内膜系统的交换速率。出芽和分裂参与高尔基体介导的囊泡运输。高尔基复合体的分裂也导致有丝分裂期间的广泛碎裂。除蛋白质因素外，膜磷脂的变化也参与了分裂。以往的研究表明，酰基辅酶A参与了高尔基体运输囊泡的分裂。在本研究中，我们提出的重要性，酰基转移酶以外的endophilin和BARS的膜动力学过程中的分裂和断裂的高尔基复合体的逆反应。当大鼠肝高尔基体膜孵育与CoA在牛血清白蛋白的存在下，大量的酰基辅酶A形成，即使在没有ATP。酰基辅酶A的形成依赖于浓度 ...更多信息 n，遵循典型的Michaelis-Menten方程（CoA的Km，100 μ M。酰基辅酶A的形成完全不依赖于ATP。在相同的条件下，游离脂肪酸不能形成酰基-CoA，这表明在此观察到的酰基-CoA的形成与在ATP存在下连接脂肪酸和CoA的酰基-CoA合成酶的合成明显不同。接下来，我们分析了溶血磷脂和磷脂在酰基辅酶A合成过程中的变化，以寻找反应的酰基供体。通常已知细胞内的溶血磷脂是低的。然而，大量的溶血磷脂酰肌醇和溶血磷脂酰胆碱产生时，肝高尔基体孵育CoA。而磷脂酰肌醇和磷脂酰胆碱的含量降低。这些结果表明，ATP非依赖性酰基辅酶A合成途径的酰基供体是膜磷脂。该活性非常高;在30分钟孵育期间，30%的磷脂酰肌醇和10%的磷脂酰胆碱转化为酰基辅酶A和相应的溶血磷脂。综上所述，我们认为溶血磷脂酰基转移酶的逆反应参与了ATP非依赖性酰基辅酶A的合成途径。溶血磷脂酰基转移酶的逆反应和正反应都是瞬时产生酰基辅酶A和溶血磷脂的过程，本文讨论了溶血磷脂酰基转移酶逆反应的生理意义。我们在HeLa细胞中表达GFP标记的GTase，高尔基体驻留蛋白，并可视化高尔基体。通过"珠加载法"使用细胞检查CoA加载的效果。当CoA被加载到细胞中时，高尔基体发生了显著的变化，CoA诱导高尔基体的快速分裂和破碎，并且在处理的1小时内发现大部分蛋白质位于内质网中。CoA的SH残基是重要的，因为碘乙酰胺处理的CoA对高尔基体没有影响。最后，我们提出酰基转移酶的逆反应改变膜的理化性质，因为溶血磷脂和脂肪酰辅酶A是水溶性的，并且逆反应是潜在的高。CoA诱导酰基转移酶的逆反应，导致促进高尔基体的分裂。磷脂和溶血磷脂加酰基辅酶A之间的可逆变化的酰基转移酶可能是重要的膜动力学的共同机制。少

The structure of the Golgi complex is totally dependent on its rates of exchange with the other inner-membrane system. The budding and fission are involved in a Golgi-mediated vesicular transport. Also the fission of the Golgi complex leads to extensive fragmentation during mitosis. In addition to proteinous factors, the changes in membrane phospholipids should be involved in the fission. Previous reports suggested that acyl-CoA was involved in the fission of the transport vesicles in Golgi apparatus. In the present study, we propose the importance of the reverse reactions of acyltransferases other than the endophilin and BARS in membrane dynamics during the fission and fragmentation of the Golgi complex.We found the ATP-independent acyl-CoA synthesis in Golgi membranes. When rat liver Golgi membranes were incubated with CoA in the presence of bovine serum albumin, large amounts of acyl-CoA were formed even in the absence of ATP. The acyl-CoA formation was dependent on the concentratio … More n of CoA and followed typical Michaelis-Menten equation (Km for CoA, 100 μM. The acyl-CoA formation was totally independent on ATP. Under the same conditions, free fatty acids failed to form acyl-CoA, suggesting that the formation of acyl-CoA observed here is clearly distinct from the synthesis by acyl-CoA synthetase that ligates fatty acid and CoA in the presence of ATP. Next, we analyzed the changes in lysophospholipids and phospholipids during the acyl-CoA synthesis to search the acyl donor of the reactions. Generally lysophospholipids within the cells are known to be low. However, large amounts of Iysophosphatidylinositol and lysophosphatidylcholine were generated when liver Golgi was incubated with CoA. In contrast, the contents of phosphatidylinositol and phosphatidylcholine were decreased. These results suggest that acyl donor of ATP-independent acyl-CoA synthetic pathway is membrane phospholipids. This activity was very high ; during 30 min-incubation, 30 % of phosphatidylinositol and 10 % of phosphatidylcholine were converted to acyl-CoA and corresponding lysophospholipids. Taken together, we proposed that the reverse reaction of lysophospholipid acyltransferases is involved in the ATP-independent acyl-CoA synthetic pathway. The reverse and forward reactions of lysophospholipid acyltransferases transiently produce acyl-CoA and lysophospholipids.We considered the physiological meaning of the reverse reaction of the acyltransferases. We expressed GFP-tagging GTase, Golgi-resident protein, in HeLa cells, and visualized Golgi apparatus. The effect of CoA loading was examined using the cells by "bead-loading method". When CoA was loaded into the cells, the Golgi was dramatically changed ; CoA induced the rapid fission and fragmentation of Golgi apparatus, and majority of the protein was found in the endoplasmic reticulum in one hr of the treatment. SH residue of CoA was important because iodeacetamide-treated CoA did not affect on the Golgi apparatus. Finally, we proposed that the reverse reaction of acyltransferases changes physiochemical propeties of membranes because lysophospholopids and fatty acyl-CoA are water-soluble, and the reverse reactions are potentially high. CoA induces the reverse reaction of acyltransferases, leading to facillitate the fission of the Golgi apparaut. The reversible change between phospholipid and lysophospholipid plus acyl-CoA by the acyltransferases may be important for the common mechanism of membrane dynamics. Less