Addison R
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd, NW, Washington, DC 20007, USA.
Fungal Genet Biol. 1998 Aug;24(3):345-53. doi: 10.1006/fgbi.1998.1066.
A translation-translocation system reconstituted with subcellular fractions from the wall-less variant fz;sg;os-1V of Neurospora crassa reproduces in vitro translocation and processing of a secretory protein. The translation extract was isolated from the wall-less variant by gently lysing cells by a freeze-thaw procedure. This method yielded more extract then the method developed previously (R. Addison, J. Biol. Chem. 262: 17031, 1987) as well as reducing microsomal contamination. The microsomal fraction was isolated from lysed cells using a series of discontinuous sucrose gradients. The resultant microsomes were less inhibitory to translation of various transcripts and consisted of a more homogenous mixture of vesicles then microsomes prepared previously. Polyclonal antibodies directed against a polypeptide of approximately 75 kDa from the microsomes were used in indirect-immunofluorescence microscopy. The resultant fluorescent pattern shows a network of tubulo-reticular structures in a juxtanuclear region, which is the pattern expected of the rough endoplasmic reticulum.
用无壁变异体粗糙脉孢菌fz;sg;os-1V的亚细胞组分重建的翻译-转运系统可在体外重现分泌蛋白的转运和加工过程。翻译提取物是通过冻融法轻柔裂解细胞，从无壁变异体中分离得到的。与之前开发的方法（R. 艾迪生，《生物化学杂志》262: 17031, 1987）相比，该方法能获得更多提取物，同时减少微粒体污染。微粒体组分是通过一系列不连续蔗糖梯度从裂解细胞中分离得到的。所得微粒体对各种转录本翻译的抑制作用较小，并且与之前制备的微粒体相比，由更均匀的囊泡混合物组成。针对微粒体中一种约75 kDa多肽的多克隆抗体用于间接免疫荧光显微镜检查。所得荧光模式显示在近核区域有一个管状网状结构网络，这是粗面内质网预期的模式。
