title: Towards delineation of a developmental α-importome in the mammalian male germline.
abstract: Nucleocytoplasmic transport mediated by importin proteins is central to many developmental processes, such as precisely regulated germ cell differentiation during spermatogenesis. Here we examine for the first time the dynamic association of importins with cargo during two successive spermatogenic stages: meiotic pachytene spermatocytes and haploid round spermatids of the adult rat testis. Immunoprecipitation followed by mass spectrometry yielded the first non-biased identification of proteins selectively interacting with importin α2, α3 and α4 in each of these cell types. Amongst the 22 novel importin binding proteins identified, 11 contain a predicted classical nuclear localization signal (cNLS) for importin α binding using a new algorithm (Kosugi et al. [22]), although only 6 of these have known nuclear functions. An importin α2-immunoprecipitated protein with a key nuclear role in meiosis, structural maintenance of chromosomes 6 (SMC6), contained a predicted bipartite NLS that was shown to be preferentially recognized by importin α together with importin β1. In contrast, the predicted cNLS of synovial sarcoma, X breakpoint 2 interacting protein (SSX2IP) was found not to confer either nuclear accumulation or direct binding to importin αs, implying that NLS prediction algorithms may identify cryptic importin binding sites or require additional refinement to increase their accuracy. Unbiased identification of importin α binding proteins in cellular differentiation represents a powerful tool to help identify the functional roles of importin αs.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Miyamoto
authors.forename: Mark A
authors.initials: MA
authors.lastname: Baker
authors.forename: Penny A
authors.initials: PA
authors.lastname: Whiley
authors.forename: Arash
authors.initials: A
authors.lastname: Arjomand
authors.forename: Justin
authors.initials: J
authors.lastname: Ludeman
authors.forename: Chin
authors.initials: C
authors.lastname: Wong
authors.forename: David A
authors.initials: DA
authors.lastname: Jans
authors.forename: Kate L
authors.initials: KL
authors.lastname: Loveland
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008297:Male; D008540:Meiosis / Q000502:physiology; D019913:Nuclear Localization Signals; D020543:Proteome / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013087:Spermatids / Q000378:metabolism*; D013090:Spermatocytes / Q000378:metabolism*; D013091:Spermatogenesis / Q000502:physiology*; D013737:Testis / Q000378:metabolism*; D028901:alpha Karyopherins / Q000378:metabolism*
chemical_list: D019913:Nuclear Localization Signals; D020543:Proteome; D011994:Recombinant Proteins; D028901:alpha Karyopherins