Elder J H, Morré D J
J Biol Chem. 1976 Aug 25;251(16):5054-68.
A fraction of intrinsic membrane proteins was prepared from the major membranous cell components of rat liver by extraction of the membranes with KCl and deoxycholate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the compositions of the intrinsic protein fractions from rough and endoplasmic reticulum, smooth endoplasmic reticulum. Golgi apparatus, plasma membrane, and nuclear envelope were similar to each other but distinct from that of mitochondria. Among endomembranes, differences were in the ratios of protein constituents plus a few protein bands of Golgi apparatus and plasma membranes not found in endoplasmic reticulum or nuclear envelope. The abilities of total rough endoplasmic reticulum, polysomes released from rough endoplasmic reticulum, and free polysomes to incorporate amino acids into the intrinsic protein fraction were tested in vitro. Polysomes bound to endoplasmic reticulum has the greatest capacity to synthesize proteins of this fraction as shown by co-purification of radioactive products and by immunoprecipitation. Although the majority of the radioactive products synthesized by bound polysomes were distinct from those synthesized by free polysomes, certain radioactive products synthesized by free polysomes also co-purified with intrinsic membrane proteins. The results show no absolute segregation between free and bound polysomes in the synthesis of intrinsic membrane proteins. However, the majority of these proteins appear to be synthesized by polysomes bound to the endoplasmic reticulum. Several intrinsic proteins found in plasma membranes do not appear in rough endoplasmic reticulum. To determine where these proteins were synthesized, the ability of other endomembrane components to support in vitro incorporation of [14C]leucine into protein was examined. In contrast to plasma membranes, isolated Golgi apparatus fractions did incorporate [14C]leucine to an extent greater than could be explained by contamination with rough endoplasmic reticulum. Golgi apparatus in situ and isolated from rat liver have polyribosomes associated with a zone of cytoplasm at the Golgi apparatus periphery occupied by tubules and vesicles. The polysomes are not directly attached to membranes as with rough endoplasmic reticulum and may represent a special class of "Golgi apparatus-associated" polysomes. The polysomes, when associated with Golgi apparatus membranes, incorporated amino acids in vitro. The products synthesized in vitro were analyzed by treatment with KCl and deoxycholate and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Certain proteins synthesized by the Golgi apparatus-associated polysomes remained insoluble after the treatment with KCl and deoxycholate. The proteins synthesized by the Golgi apparatus fraction had mobilities similar to proteins in plasma membranes which were absent from endoplasmic reticulum, and which were relatively minor components of Golgi apparatus...
通过用氯化钾和脱氧胆酸盐提取大鼠肝脏主要膜性细胞成分，制备了一部分内在膜蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明，来自糙面内质网、光面内质网、高尔基体、质膜和核被膜的内在蛋白组分的组成彼此相似，但与线粒体的不同。在内膜系统中，差异在于蛋白质成分的比例以及高尔基体和质膜中一些在内质网或核被膜中未发现的蛋白条带。在体外测试了总糙面内质网、从糙面内质网释放的多核糖体和游离多核糖体将氨基酸掺入内在蛋白组分的能力。如放射性产物的共纯化和免疫沉淀所示，与内质网结合的多核糖体合成该组分蛋白质的能力最强。尽管结合多核糖体合成的大多数放射性产物与游离多核糖体合成的不同，但游离多核糖体合成的某些放射性产物也与内在膜蛋白共纯化。结果表明，在内在膜蛋白的合成中，游离多核糖体和结合多核糖体之间没有绝对的分离。然而，这些蛋白中的大多数似乎是由与内质网结合的多核糖体合成的。质膜中发现的几种内在蛋白在内质网中未出现。为了确定这些蛋白在哪里合成，研究了其他内膜成分支持体外将[14C]亮氨酸掺入蛋白质的能力。与质膜不同，分离的高尔基体组分确实掺入了[14C]亮氨酸，其掺入程度大于因糙面内质网污染所能解释的程度。原位和从大鼠肝脏分离的高尔基体在高尔基体周边被小管和小泡占据的细胞质区域有与多核糖体相关联。这些多核糖体不像糙面内质网那样直接附着在膜上，可能代表一类特殊的“高尔基体相关”多核糖体。当与高尔基体膜相关联时，这些多核糖体在体外掺入氨基酸。体外合成的产物经氯化钾和脱氧胆酸盐处理后进行分析，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离。高尔基体相关多核糖体合成的某些蛋白质在经氯化钾和脱氧胆酸盐处理后仍不溶。高尔基体组分合成的蛋白质的迁移率与内质网中不存在、在高尔基体中相对含量较少的质膜中的蛋白质相似……
