Identification of Karyopherin α1 and α7 Interacting Proteins in Porcine Tissue

Abstract

Specialized trafficking systems in eukaryotic cells serve a critical role in partitioning intracellular proteins between the nucleus and cytoplasm. Cytoplasmic proteins (including chromatin remodeling enzymes and transcription factors) must gain access to the nucleus to exert their functions to properly program fundamental cellular events ranging from cell cycle progression to gene transcription. Knowing that nuclear import mediated by members of the karyopherin α family of transport receptors plays a critical role in regulating development and differentiation, we wanted to determine the identity of proteins that are trafficked by this karyopherin α pathway. To this end, we performed a GST pull-down assay using porcine orthologs of karyopherin α1 (KPNA1) and karyopherin α7 (KPNA7) and prey protein derived from porcine fibroblast cells and used a liquid chromatography and tandem mass spectrometry (LC-MS/MS) approach to determine the identity of KPNA1 and KPNA7 interacting proteins. Our screen revealed that the proteins that interact with KPNA1 and KPNA7 are generally nuclear proteins that possess nuclear localization signals. We further validated two candidate proteins from this screen and showed that they are able to be imported into the nucleus in vivo and also interact with members of the karyopherin α family of proteins in vitro. Our results also reveal the utility of using a GST pull-down approach coupled with LC-MS/MS to screen for protein interaction partners in a non-traditional model system.

Functional characterization of importin α8 as a classical nuclear localization signal receptor

“…Bacterially produced recombinant importin α8 exhibited strong binding to importin β1, consistent with previous reports [15][16][17]. In addition, we demonstrated that importin α8 interacted with the SV40TNLS substrate and transported it to the nucleus.…”

“…Previous reports indicated that importin α8 only binds weakly, or not at all, to the SV40TNLS [16][17][18]. However, amino acid sequence alignment of importin α8 and importin α1 revealed that the critical amino acids in the major and minor NLS-binding sites are conserved ( Fig.…”

“…Importin α8 was shown to bind to importin β1 [15][16][17] and to cNLSs from several karyophilic proteins, including nucleoplasmin 2, retinoblastoma, and sperm associated antigen 17-like protein, although binding to the SV40TNLS either did not occur or was very weak [16][17][18]. Despite these data, and the structural similarities between importin α8 and other cNLS receptors, there is no experimental evidence that importin α8 actually functions as one.…”

“…Importin α8 is the most recently identified importin α family member in a number of species [14][15][16][17][18][19]. Importin α8 is expressed in oocytes and embryos [14,15,18], and importin α8…”

Identification of Cargo Proteins Specific for Importin-β with Importin-α Applying a Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based in Vitro Transport System*

“…Screening synthetic libraries, such as in vitro virus libraries (18) and oriented peptide libraries (19), is effective for identifying amino acid sequences that bind to Imp-␣ and act as cNLSs, but these methods do not identify native cargoes. The identification of native Imp-␣ cargoes was performed using Far Western-based cellular proteomic approaches (20), yeast two-hybrid screening (21), GST pulldown assays (22,23), and co-immunoprecipitation techniques (24). These methods depend on the protein-protein interaction between Imp-␣ and its cargo.…”