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.

Figure 1. PAGE analysis of KPNA1 and KPNA7 GST pull-down reactions.

A representative image of the resolved protein eluate following the GST pull-down assay. Lanes are labeled as follows: Lane 1, cleaned prey protein input (not eluted from GST column); Lane 2, GST and prey protein; Lane 3, GST alone; Lane 4, GST-KPNA7 and prey; Lane 5, GST-KPNA7 alone; Lane 6, GST-KPNA1 and prey; Lane 7, KPNA1 alone. Arrows indicate location of bands that were excised for proteomic analysis.

Figure 2. Recombinant GST-SP17 and GST-RREB adopt a nuclear localization.

Fluorescein-labeled GST-SP17 and GST-RREB adopt a nuclear localization in porcine oocytes and cleavage stage embryos, while fluorescein-labeled GST does not adopt a predominant nuclear location at these developmental stages. Panels A-E reflect oocytes and embryos co-injected with fluorescein-labeled GST-SP17 and Alexa594-labeled GST-NLS; panels F-G reflect oocytes and embryos co-injected with fluorescein-labeled GST-RREB and Alexa594-labeled GST-NLS; panels H-L reflect oocytes and embryos co-injected with fluorescein-labeled GST and Alexa594-labeled GST-NLS. In all cases panels beginning with the same letter are derived from the same representative oocyte or embryo; images were captured as an optical section using confocal microscopy. DNA is shown in panels A1–L1; fluorescein-labeled proteins are shown in panels A2–L2, Alexa594-labeled proteins are shown in panels A3–L3. A GV-stage oocyte (GV), pronuclear stage embryo (PN), 2-cell stage embryo (2-cell), 4-cell stage embryo (4-cell) and 8-cell stage embryo (8-cell) are shown as indicated in the figure.

Figure 3. Nuclear accumulation of GST-SP17 and GST-RREB is time-dependent.

Panels A-C reflect oocytes co-injected with fluorescein-labeled GST-SP17 and Alexa594-labeled GST-NLS; panels D-F reflect oocytes co-injected with fluorescein-labeled GST-RREB and Alexa594-labeled GST-NLS; panels G-I reflect oocytes co-injected with fluorescein-labeled GST and Alexa594-labeled GST-NLS. In all cases panels beginning with the same letter are derived from the same representative oocyte; images were captured as an optical section using confocal microscopy. DNA is shown in panels A1–I1; fluorescein-labeled proteins are shown in panels A2–I2, Alexa594-labeled proteins are shown in panels A3–I3. Both GST-SP17 and GST-RREB show a weak nuclear accumulation 30 minutes after microinjection into GV stage oocytes. GST-SP17 and GST-RREB both show an increased presence in the nucleus at 3 and 6 hours after microinjection. In contrast, the co-injected GST-NLS adopts a nuclear localization rapidly after microinjection and has a profound nuclear accumulation 30 minutes following microinjection. Fluorescein-labeled GST remains a predominantly cytoplasmic protein at all time-points following microinjection.

Figure 4. Recombinant GST-SP17 and GST-RREB interact with multiple karyopherin α subtypes.

S³⁵-labeled versions of porcine KPNA1, KPNA2, KPNA3, KPNA5, KPNA6, and KPNA7 were tested for their ability to interact with GST, GST-SP17, GST-RREB, GST-NLS, and a GST tagged version of karyopherin β (KPNB). Shown in this figure are the respective radiographs of this in vitro binding assay. Lanes are labeled as follows: GST (recombinant GST); SP17 (GST-SP17); RREB (GST-RREB); NLS (GST-NLS); KPNB (GST-KPNB), and lysate (crude lysate from the respective S³⁵-labeled KPNA in vitro translation reactions, corresponding to 10% of input into each binding reaction). The intensity of the radiolabeled bands was quantified for each karyopherin α subtype; band intensity normalized to the intensity of binding to KPNB (100%) for KPNA1, KPNA2, KPNA3, KPNA5, and KPNA7. *Because the KPNA6 construct lacked the majority of the importin β binding domain, little interaction with KPNB was observed and in this case KPNA6 intensity was normalized to lysate input, rather than KPNB binding.