The α-importome of mammalian germ cell maturation provides novel insights for importin biology.
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.
Department of Biochemistry and Molecular Biology and Australian Research Council Centre of Excellence in Biotechnology and Development, Canberra, Australian Capital Territory, Australia; and.
Arash
A
Arjomand
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.
Mark A
MA
Baker
Department of Biochemistry and Molecular Biology and.
Chen
C
Li
Department of Biochemistry and Molecular Biology and.
Ashley M
AM
Buckle
Department of Biochemistry and Molecular Biology and Australian Research Council Centre of Excellence in Biotechnology and Development, Canberra, Australian Capital Territory, Australia; and.
David A
DA
Jans
Department of Biochemistry and Molecular Biology and Department of Anatomy and Developmental Biology, School of Biological Sciences, Monash University, Clayton, Victoria, Australia; Australian Research Council Centre of Excellence in Biotechnology and Development, Canberra, Australian Capital Territory, Australia; and kate.loveland@monash.edu.
Kate L
KL
Loveland
Department of Biochemistry and Molecular Biology and.
Yoichi
Y
Miyamoto
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry; D002451:Cell Compartmentation; D004265:DNA Helicases / Q000737:chemistry; D008297:Male; D008540:Meiosis; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry; D049471:Pachytene Stage; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000502:physiology; D017434:Protein Structure, Tertiary; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013087:Spermatids / Q000378:metabolism / Q000648:ultrastructure; D013091:Spermatogenesis / Q000502:physiology*; D014157:Transcription Factors / Q000737:chemistry; D028901:alpha Karyopherins / Q000032:analysis / Q000502:physiology*; D028961:beta Karyopherins / Q000032:analysis / Q000502:physiology
D002352:Carrier Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C000592120:Setx protein, rat; D014157:Transcription Factors; D028901:alpha Karyopherins; D028961:beta Karyopherins; C578929:Smarca4 protein, rat; D004265:DNA Helicases
mass spectrometry; nuclear localization signal; nuclear transport; spermatogenesis