Towards delineation of a developmental α-importome in the mammalian male germline
| Autor: | Justin P. Ludeman, Penelope Alexandra Falshaw Whiley, Mark Baker, Kate L Loveland, Yoichi Miyamoto, Arash Arjomand, Chin Long Wong, David A. Jans |
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| Rok vydání: | 2012 |
| Předmět: |
Male
alpha Karyopherins animal structures Proteome Immunoprecipitation Cellular differentiation Blotting Western Nuclear Localization Signals Importin α Active Transport Cell Nucleus NLS Enzyme-Linked Immunosorbent Assay Importin Biology DNA-binding protein environment and public health SMC6 Immunoenzyme Techniques Rats Sprague-Dawley 03 medical and health sciences 0302 clinical medicine Spermatocytes Testis Animals Binding site Spermatogenesis Molecular Biology 030304 developmental biology Genetics 0303 health sciences Cell Biology Spermatids Recombinant Proteins Cell biology Rats Meiosis 030220 oncology & carcinogenesis Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization embryonic structures Nuclear transport Nuclear localization sequence |
| Zdroj: | Biochimica et biophysica acta. 1833(3) |
| ISSN: | 0006-3002 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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