The major yolk protein of sea urchins is endocytosed by a dynamin-dependent mechanism
The major yolk protein of sea urchins is endocytosed by a dynamin-dependent mechanism
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DOI：
10.1095/biolreprod.104.027730
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发表时间：
2004-09-01
影响因子：
3.6
通讯作者：
Wessel, GM
中科院分区：
文献类型：
--
作者：
Brooks, JM;Wessel, GM
在卵子发生过程中，海胆卵母细胞通过合成和输入特定的蛋白质库来驱动受精和早期胚胎发生，从而生长到其原始大小的10倍。在卵黄发生生长期间，主要卵黄蛋白（MYP），一种转铁蛋白样蛋白，在肠道中合成，运输到卵巢，并被卵母细胞积极内吞。在这里，我们开始剖析这一机制，首先测试的假设，MYP的内吞作用是动力蛋白依赖。我们已经确定了一个海胆发动蛋白的cDNA，是高度相似的氨基酸序列，结构和大小的哺乳动物发动蛋白I：它包含一个N-末端GT3结构域，pleckstrin同源结构域，和C-末端脯氨酸丰富的结构域。海胆动力蛋白在卵母细胞的皮质富集，并在卵母细胞外周与MYP内吞囊泡共定位。为了测试MYP内吞作用和发动蛋白之间的功能关系，我们使用了一种显性阴性的人发动蛋白I突变蛋白，该突变蛋白在GTdR结构域（hDyn（K44 A））内含有改变，以特异性地竞争发动蛋白功能。使用荧光MYP构建体，以遵循其内吞作用，以及一般的内吞标记，我们证明，动力蛋白功能的破坏显着降低MYP的摄取，但不影响液相内吞作用。使用这种特定的生物化学方法，我们能够分离卵子发生过程中的内吞作用的不同途径，并了解到动力蛋白介导的内吞作用是负责MYP内吞作用，但不是液相摄取。
Sea urchin oocytes grow to 10 times their original size during oogenesis by both synthesizing and importing a specific repertoire of proteins to drive fertilization and early embryogenesis. During the vitellogenic growth period, the major yolk protein (MYP), a transferrin-like protein, is synthesized in the gut, transported into the ovary, and actively endocytosed by the oocytes. Here, we begin to dissect this mechanism by first testing the hypothesis that MYP endocytosis is dynamin-dependent. We have identified a sea urchin dynamin cDNA that is highly similar in amino acid sequence, structure, and size to mammalian dynamin I: it contains an N-terminal GTPase domain, a pleckstrin-homology domain, and a C-terminal proline-rich domain. Sea urchin dynamin is enriched at the cortex of oocytes and colocalizes to MYP endocytic vesicles at the oocyte periphery. To test for a functional relationship between MYP endocytosis and dynamin, we used a dominant-negative human dynamin I mutant protein containing an alteration within the GTPase domain (hDyn(K44A)) to specifically compete for dynamin function. Using a fluorescent MYP construct to follow its endocytosis solely, as well as a general endocytosis marker, we demonstrate that the disruption of dynamin function significantly reduces MYP uptake but does not affect fluid-phase endocytosis. Using this specific biochemical approach, we are able to separate distinct pathways of endocytosis during oogenesis and learn that dynamin-mediated endocytosis is responsible for MYP endocytosis but not fluid-phase uptake.