Rat Spag5 associates in somatic cells with endoplasmic reticulum and microtubules but in spermatozoa with outer dense fibers
Rat Spag5 associates in somatic cells with endoplasmic reticulum and microtubules but in spermatozoa with outer dense fibers
复制标题
DOI：
10.1002/mrd.20388
复制
发表时间：
2006-01-01
影响因子：
2.5
通讯作者：
van der Hoorn, FA
中科院分区：
文献类型：
--
作者：
Fitzgerald, CJ;Oko, RJ;van der Hoorn, FA
亮氨酸拉链基序被认为是一种重要而特异的相互作用基序，被定位于外部致密纤维的各种精子尾部蛋白所使用。我们已经发现，大鼠Odf1，一个主要的整体ODF蛋白，利用其亮氨酸拉链与Odf2，另一个主要的ODF蛋白，Spag4，定位于ODF和轴丝微管双联体之间的界面，和Spag5。大鼠Spag5序列表明与人类Astrin的密切关系，Astrin是一种微管结合纺锤体蛋白，表明Spag5和Spag4一样，可能与精子尾部轴丝相关。RT PCR分析表明Spag5在各种组织和体细胞中的表达Spag5定位于内质网和微管，如预期的Astrin直向同源物。MT结合在体内和体外MT结合测定中均得到证实：体细胞含有58 kDa MT相关Spag5蛋白。大鼠体细胞和雄性生殖细胞在不同的发展阶段，使用抗Spag5抗体的蛋白质印迹分析表明，在精子发生过程中的蛋白质表达模式的变化，精子尾部含有一个58 kDa的Spag5蛋白。使用亲和纯化的抗Spag5抗体的免疫电子显微镜显示，在大鼠细长精子细胞和附睾精子的Spag5蛋白与ODF，但不与轴丝MT。这一观察结果与另一种ODF 1结合蛋白MT结合蛋白Spag4的观察结果相反，Spag4存在于ODF和轴丝之间。我们的数据表明Spag5在体细胞和雄性生殖细胞中具有不同的定位，这表明可能具有不同的功能。
The leucine zipper motif has been identified as an important and specific interaction motif used by various sperm tail proteins that localize to the outer dense fibers. We had found that rat Odf1, a major integral ODF protein, utilizes its leucine zipper to associate with Odf2, another major ODF protein, Spag4 which localizes to the interface between ODF and axonemal microtubule doublets, and Spag5. The rat Spag5 sequence indicated a close relationship with human Astrin, a microtubule-binding spindle protein suggesting that Spag5, like Spag4, may associate with the sperm tail axoneme. RT PCR assays indicated expression of Spag5 in various tissues and in somatic cells Spag5 localizes to endoplasmic reticulum and microtubules, as expected for an Astrin orthologue. MT binding was confirmed both in vivo and in in vitro MT-binding assays: somatic cells contain a 58 kDa MT-associated Spag5 protein. Western blotting assays of rat somatic cells and male germ cells at different stages of development using anti-Spag5 antibodies demonstrated that the protein expression pattern changes during spermatogenesis and that sperm tails contain a 58 kDa Spag5 protein. Use of affinity-purified anti-Spag5 antibodies in immuno electron microscopy shows that in rat elongated spermatids and epididymal sperm the Spag5 protein associates with ODF, but not with the axonemal MTs. This observation is in contrast to that for the other Odf1-binding, MT-binding protein Spag4, which is present between ODF and axoneme. Our data demonstrate that Spag5 has different localization in somatic versus male germ cells suggesting the possibility of different function.