Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation.
Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation.
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DOI：
10.1083/jcb.107.2.413
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发表时间：
1988-08
期刊：
影响因子：
--
通讯作者：
Lodish HF
中科院分区：
文献类型：
--
作者：
Lehnert ME;Lodish HF
鼠红白血病 (MEL) 细胞是研究红系分化过程中细胞骨架生物发生的一个有价值的系统。当附着到纤连蛋白包被的培养皿上时，MEL 细胞在添加 DMSO 后诱导 7 天的分化过程，在此期间它们去核并达到网织红细胞阶段 (Patel, V. P. 和 H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118)；它们积累的带 3、血影蛋白和锚蛋白的数量与成熟红细胞中的数量相当。为了跟踪分化过程中血影蛋白的生物合成，用[35S]蛋氨酸代谢标记的细胞的膜和细胞骨架蛋白通过SDS溶解，并通过特异性免疫吸附回收α和β血影蛋白。在未诱导和 3 d 诱导细胞中，α/β 血影蛋白的相对合成比例约为 1:3。在未诱导的 MEL 细胞中，新合成的 α 和 β 血影蛋白被降解，半衰期相似，约为 10 小时。相比之下，在 3 d 分化的 MEL 细胞中，新产生的 β 血影蛋白比 α 血影蛋白更不稳定； α 和 β 血影蛋白链的半衰期分别约为 22 和 8 小时。因此，等量的α和β血影蛋白的积累是由不等的合成和不等的降解引起的。根据 Northern 印迹分析判断，肌动蛋白 mRNA 水平在整个 7 天分化期间相对恒定。 α 和 β 血影蛋白 mRNA 在未诱导的细胞中几乎检测不到，在诱导的前 4 天内增加，此后保持恒定。相比之下，条带 3 mRNA 在分化第 4 天首次被检测到。因此，在去核网织红细胞中积累的大部分血影蛋白是在红细胞生成的最后几天合成的，伴随着带3合成的开始。为了确定这是否发生在正常小鼠红细胞生成中，我们分析了单次注射[35S]甲硫氨酸后成熟红细胞中标记膜蛋白的出现率。我们的结果表明，成熟红细胞中的大多数血影蛋白和带 3 是在骨髓红细胞生成的最后几天合成的，并且在骨髓中，带 3 和蛋白 4.1 的合成时间比 α 和 β 血影蛋白、锚蛋白和肌动蛋白稍晚。
Murine erythroleukemia (MEL) cells represent a valuable system to study the biogenesis of the cytoskeleton during erythroid differentiation. When attached to fibronectin-coated dishes MEL cells induce, upon addition of DMSO, a 7-d differentiation process during which they enucleate and reach the reticulocyte stage (Patel, V. P., and H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118); they accumulate band 3, spectrin, and ankyrin in amounts equivalent to those found in mature red blood cells. To follow the biosynthesis of spectrin during differentiation, membranes and cytoskeletal proteins of cells metabolically labeled with [35S]methionine were solubilized by SDS and alpha and beta spectrins were recovered by specific immunoadsorption. In both uninduced and 3-d induced cells, the relative synthesis of alpha/beta spectrin is approximately 1:3. In uninduced MEL cells newly synthesized alpha and beta spectrins are degraded with a similar half- life of approximately 10 h. In contrast, in 3-d differentiated MEL cells newly made beta spectrin is much more unstable than alpha spectrin; the half-lives of alpha and beta spectrin chains are approximately 22 and 8 h, respectively. Thus, accumulation of equal amounts of alpha and beta spectrin is caused by unequal synthesis and unequal degradation. As judged by Northern blot analyses, the level of actin mRNA is relatively constant throughout the 7-d differentiation period. alpha and beta spectrin mRNAs are barely detectable in uninduced cells, increase during the first 4 d of induction, and remain constant thereafter. In contrast, band 3 mRNA is first detectable on day 4 of differentiation. Thus, most of the spectrin that accumulates in enucleating reticulocytes is synthesized during the last few days of erythropoiesis, concomitant with the onset of band 3 synthesis. To determine whether this was occurring in normal mouse erythropoiesis, we analyzed the rate of appearance of labeled membrane proteins in mature erythrocytes after a single injection of [35S]methionine. Our results show that most of the spectrin and band 3 in mature erythrocytes is synthesized during the last days of bone marrow erythropoiesis, and that, in the marrow, band 3 and protein 4.1 are synthesized at a somewhat later stage of development than are alpha and beta spectrin, ankyrin, and actin.