Characterization of the Golgi complex cleared of proteins in transit and examination of calcium uptake activities
Characterization of the Golgi complex cleared of proteins in transit and examination of calcium uptake activities
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DOI：
10.1091/mbc.8.10.1911
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发表时间：
1997-10-01
影响因子：
3.3
通讯作者：
Howell, KE
中科院分区：
文献类型：
--
作者：
Taylor, RS;Jones, SM;Howell, KE
为了表征内源性分子和高尔基复合体的活性，用环己亚胺（CHX）处理大鼠肝细胞清除了运输中的蛋白质b> 99%。运输过程中蛋白质的损失导致高尔基池和堆栈的凝结。无论是否有转运蛋白[对照物（CTL SGF1）和环己亚胺（CHX SGF1）]，分离堆叠的高尔基体馏分都是同样有效的。电镜和形态计量学分析表明，其中90%以上的元素可被鉴定为高尔基堆或池。生化分析表明，在核后上清液中，顺式、中式、反式和TGN高尔基体标记物富集200- 400倍，不含转运蛋白时富集400- 700倍。为了提供分泌途径后期所需钙进口机制的信息，研究了钙摄取到CTL SGF1和CHX SGF1。所有进入CTL SGF1的钙摄取都依赖于一个非高尔基复合体的抗塔素泵和一个高尔基体的塔素敏感泵。使用CHX SGF1进行的实验表明，抗塔素活性是转运到质膜的质膜钙atp酶异构体，而塔素敏感泵是肌浆/内质网钙atp酶异构体。在体内，这两种钙三磷酸腺苷酶的作用都是维持高尔基腔内的毫摩尔钙水平。
To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were > 99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that > 90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.