关键词:
nuclear transport
spermatogenesis
mass spectrometry
nuclear localization signal
NUCLEAR-LOCALIZATION SIGNAL
NUCLEOCYTOPLASMIC TRANSPORT
KARYOPHERIN ALPHA
IDENTIFICATION
RECOGNITION
EXPRESSION
PROTEINS
EXPORT
摘要:
Importin alpha proteins function as adaptors to connect a cargo protein and importin beta 1 in the classical nuclear import pathway. Here we measure for the first time the stoichiometry of importins alpha 2, alpha 3, alpha 4, and beta 1 in primary cells corresponding to 2 successive stages of rat spermatogenesis: meiotic spermatocytes and haploid round spermatids. Importin alpha 2 levels were more than 2-fold higher in spermatocytes than in spermatids, while importins alpha 4 and beta 1 levels did not differ significantly. We performed a comprehensive proteomics analysis to identify binding proteins in spermatocytes and spermatids using recombinant importin alpha 2 and alpha 4 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 alpha through the conserved C-terminal acidic domain (ARM repeats 9-10), and provided functional validation of a predicted importin alpha 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 alpha s may serve unique and nonredundant roles in mediating cellular differentiation.