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

高尔基络合物的结构完全取决于它与其他细胞膜系统的交换速率。出芽和裂变参与高尔基介导的囊泡运输。在有丝分裂过程中，高尔基复合体的裂变也会导致广泛的分裂。除蛋白质因子外，膜磷脂的变化应与裂变有关。以前的报道认为酰基辅酶a参与了高尔基体运输囊泡的裂变。在本研究中，我们提出了在高尔基复合体裂变和断裂过程中，除亲内蛋白和BARS外，酰基转移酶的逆反应在膜动力学中的重要性。我们在高尔基膜中发现了atp非依赖性酰基辅酶a的合成。在牛血清白蛋白存在的情况下，用CoA孵育大鼠肝脏高尔基膜，即使在没有ATP的情况下，也能形成大量的酰基CoA。酰基-CoA的形成与CoA的浓度有关，遵循典型的Michaelis-Menten方程（Km for CoA, 100 μM）。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辅酶a，这表明这里观察到的酰基辅酶a的形成与ATP存在下连接脂肪酸和辅酶a的酰基辅酶a合成酶的合成明显不同。接下来，我们分析了在酰基辅酶a合成过程中溶血磷脂和磷脂的变化，以寻找反应的酰基供体。通常已知细胞内的溶血磷脂较低。然而，当肝脏高尔基体与CoA孵育时，产生大量的溶血磷脂酰肌醇和溶血磷脂酰胆碱。相反，磷脂酰肌醇和磷脂酰胆碱含量降低。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。这个活动非常高；孵育30 min后，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，我们提出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合成途径。溶血磷脂酰基转移酶的逆反应和正向反应短暂地产生酰基辅酶a和溶血磷脂。我们考虑了酰基转移酶逆反应的生理意义。我们在HeLa细胞中表达gfp标记的高尔基驻留蛋白GTase，并可视化高尔基体。用“珠载法”检测细胞对CoA的加载效果。当辅酶a被装载到细胞中时，高尔基体发生了巨大的变化；CoA诱导高尔基体快速分裂和断裂，大部分蛋白在处理后1小时内质网中发现。辅酶a的SH残留是重要的，因为碘乙酰胺处理的辅酶a不影响高尔基体。最后，我们提出酰基转移酶的逆反应改变了膜的物理化学性质，因为溶血磷脂和脂肪酰基辅酶a是水溶性的，并且逆反应可能很高。辅酶a诱导酰基转移酶的逆反应，从而促进高尔基体的裂变。磷脂和溶血磷脂加酰基辅酶a在酰基转移酶作用下的可逆变化可能对膜动力学的共同机制有重要意义。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