Arjomand Arash, Baker Mark A, Li Chen, Buckle Ashley M, Jans David A, Loveland Kate L, Miyamoto Yoichi
Department of Biochemistry and Molecular Biology and Australian Research Council Centre of Excellence in Biotechnology and Development, Canberra, Australian Capital Territory, Australia; and.
Australian Research Council Centre of Excellence in Biotechnology and Development, Canberra, Australian Capital Territory, Australia; and Priority Research Centre in Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia.
FASEB J. 2014 Aug;28(8):3480-93. doi: 10.1096/fj.13-244913. Epub 2014 Apr 30.
Importin α proteins function as adaptors to connect a cargo protein and importin β1 in the classical nuclear import pathway. Here we measure for the first time the stoichiometry of importins α2, α3, α4, and β1 in primary cells corresponding to 2 successive stages of rat spermatogenesis: meiotic spermatocytes and haploid round spermatids. Importin α2 levels were more than 2-fold higher in spermatocytes than in spermatids, while importins α4 and β1 levels did not differ significantly. We performed a comprehensive proteomics analysis to identify binding proteins in spermatocytes and spermatids using recombinant importin α2 and α4 proteins. Among the 100 candidate partners, 42 contained a strong classical nuclear localization signal (cNLS; score of>6 by cNLS Mapper), while 8 nuclear proteins lacked any cNLS. In addition, we developed a new strategy to predict which cargoes bind to importin α through the conserved C-terminal acidic domain (ARM repeats 9-10), and provided functional validation of a predicted importin α C-terminal binding segment in Senataxin and Smarca4. Evaluation of this set of candidate binding partners from spermatogenic cells using several bioinformatics approaches provides new evidence that individual importin αs may serve unique and nonredundant roles in mediating cellular differentiation.
输入蛋白α家族蛋白在经典的核输入途径中作为衔接蛋白，连接货物蛋白与输入蛋白β1。在此，我们首次测定了大鼠精子发生连续两个阶段（减数分裂期精母细胞和单倍体圆形精子细胞）的原代细胞中输入蛋白α2、α3、α4和β1的化学计量。精母细胞中的输入蛋白α2水平比精子细胞中的高2倍多，而输入蛋白α4和β1的水平无显著差异。我们进行了一项全面的蛋白质组学分析，以使用重组输入蛋白α2和α4蛋白鉴定精母细胞和精子细胞中的结合蛋白。在100个候选伴侣中，42个含有强经典核定位信号（cNLS；cNLS Mapper评分>6），而8个核蛋白缺乏任何cNLS。此外，我们开发了一种新策略，以预测哪些货物通过保守的C端酸性结构域（ARM重复序列9-10）与输入蛋白α结合，并对Senataxin和Smarca4中预测的输入蛋白α C端结合片段进行了功能验证。使用几种生物信息学方法对这组来自生精细胞的候选结合伴侣进行评估，提供了新的证据，表明单个输入蛋白α在介导细胞分化中可能发挥独特且非冗余的作用。
