The steady-state distribution of glycosyltransferases between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10
The steady-state distribution of glycosyltransferases between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10
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DOI：
10.1111/j.1600-0854.2005.00333.x
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发表时间：
2005-11-01
期刊：
影响因子：
4.5
通讯作者：
Storrie, B
中科院分区：
文献类型：
--
作者：
Rhee, SW;Starr, T;Storrie, B
一些证据支持高尔基蛋白驻留的新模型，其中这些蛋白质在高尔基体和内质网（ER）之间循环。然而，为了保持两种细胞器之间的功能区别，这种er驻留高尔基酶池必须很小。我们量化了HeLa细胞中两种高尔基糖基转移酶的分布来验证这一预测。我们推断，最佳实践的定量解决方案将来自于将图像视为数据数组而不是图像。通过反卷积和计算机计算细胞器边界，荧光显微镜下内源性β 1,4-半乳糖基转移酶和udp - n -乙酰半乳糖胺：多肽n -乙酰半乳糖基转移酶2与绿色荧光蛋白（GFP）融合的高尔基分数为91%。免疫金标记，然后电镜和模型分析得出类似的值。数值反映稳态条件，因为包含蛋白质合成抑制剂没有影响。这些数据强烈表明，GFP嵌合体与细胞器蛋白的荧光可以作为蛋白质分布的有效指标，更普遍的是，荧光显微镜可以为蛋白质定量提供一种有效、快速的方法。总之，我们发现循环高尔基糖基转移酶的内质网池很小，大约是高尔基体浓度的1/100。
Several lines of evidence support a novel model for Golgi protein residency in which these proteins cycle between the Golgi apparatus and the endoplasmic reticulum (ER). However, to preserve the functional distinction between the two organelles, this pool of ER-resident Golgi enzymes must be small. We quantified the distribution for two Golgi glycosyltransferases in HeLa cells to test this prediction. We reasoned that best-practice, quantitative solutions would come from treating images as data arrays rather than pictures. Using deconvolution and computer calculated organellar boundaries, the Golgi fraction for both endogenous beta 1,4-galactosyltransferase and UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase 2 fused with green fluorescent protein (GFP) was 91% by fluorescence microscopy. Immunogold labeling followed by electron microscopy and model analysis yielded a similar value. Values reflect steady-state conditions, as inclusion of a protein synthesis inhibitor had no effect. These data strongly suggest that the fluorescence of a GFP chimera with an organellar protein can be a valid indicator of protein distribution and more generally that fluorescent microscopy can provide a valid, rapid approach for protein quantification. In conclusion, we find the ER pool of cycling Golgi glycosyltransferases is small and approximately 1/100 the concentration found in the Golgi apparatus.