Rodriguez M, Moreau P, Paulik M, Lawrence J, Morré D J, Morré D
Department of Foods and Nutrition, Purdue University, West Lafayette, IN.
Biochim Biophys Acta. 1992 Jun 11;1107(1):131-8. doi: 10.1016/0005-2736(92)90338-m.
This report concerns development of a cell-free system from rat liver to study transport of membrane constituents from the Golgi apparatus to the plasma membrane. Highly purified Golgi apparatus as donor and a mixture of sheets and vesicles as plasma membrane acceptor fractions were combined to analyze requirements for lipid and protein transport. In the reconstituted system, the Golgi apparatus donor was in suspension. To measure transfer, membrane constituents of the donor membranes were radiolabeled with [3H]acetate (lipids) or [3H]leucine (proteins). The plasma membrane vesicles were used as the acceptor and were unlabeled and immobilized on nitrocellulose for ease of recovery and analysis. The reconstituted cell-free transfer was dependent on temperature, but even at 37 degrees C, the amount of transfer did not increase with added ATP, was not specific for any particular membrane fraction or subfraction nor was it facilitated by cytosol. ATP was without effect both in the presence or absence of a cytosolic fraction capable of the support of cell-free transfer in other systems. In contrast to results with ATP, NADH added to the reconstituted system resulted in an increased amount of transfer. A further increase in transfer was obtained with NADH plus a mixture of ascorbate and dehydroascorbate to generate ascorbate free radical. The transfer of labeled membrane constituents from the Golgi apparatus to the plasma membrane supported by NADH plus ascorbate radical was stimulated by a cytosol fraction enriched in less than 10 kDa components. This was without effect in the absence of NADH/ascorbate radical or with ATP as the energy source. Specific transfer was inhibited by both N-ethylmaleimide and GTP gamma S. The findings point to the possibility of redox activities associated with the trans region of the Golgi apparatus as potentially involved in the transport of membrane vesicles from the Golgi apparatus to the cytoplasmic surface of the plasma membrane.
本报告涉及大鼠肝脏无细胞体系的开发，用于研究膜成分从高尔基体向质膜的转运。将高度纯化的高尔基体作为供体，以及片状物和囊泡的混合物作为质膜受体组分相结合，以分析脂质和蛋白质转运的需求。在重构体系中，高尔基体供体处于悬浮状态。为了测量转运，供体膜的膜成分用[3H]乙酸盐（脂质）或[3H]亮氨酸（蛋白质）进行放射性标记。质膜囊泡用作受体，未标记并固定在硝酸纤维素上，以便于回收和分析。重构的无细胞转运依赖于温度，但即使在37℃时，转运量也不会随着添加ATP而增加，对任何特定的膜组分或亚组分都没有特异性，也不会被胞质溶胶促进。无论是否存在能够在其他体系中支持无细胞转运的胞质组分，ATP都没有作用。与ATP的结果相反，添加到重构体系中的NADH导致转运量增加。当NADH加上抗坏血酸盐和脱氢抗坏血酸盐的混合物以产生抗坏血酸自由基时，转运进一步增加。由NADH加上抗坏血酸自由基支持的从高尔基体到质膜的标记膜成分的转运受到富含小于10 kDa组分的胞质溶胶组分的刺激。在没有NADH/抗坏血酸自由基或使用ATP作为能量来源的情况下，这没有作用。特异性转运受到N-乙基马来酰亚胺和GTPγS的抑制。这些发现表明，与高尔基体反式区域相关的氧化还原活性有可能参与从高尔基体到质膜胞质表面的膜囊泡转运。
