Regulation of Nuclear Import and Export of Negative Cofactor 2

Abstract

The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2␣ and NC2␤, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2␣ and NC2␤ depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin ␣/␤ heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as cotransfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2␤. In contrast, NC2␣ does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2␣ masks the NES in NC2␤, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin ␣/␤-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits. The negative cofactor 2 (NC2)2 is a protein complex composed of two subunits, NC2␣ (DRAP1) and NC2␤ (Dr1). Both subunits are conserved in eukaryotes and essential for Saccharomyces cerevisae viability (1, 2). NC2␣ and NC2␤ heterodimerize via histone-fold domains and associate with the promotor-bound TATA-binding protein (3, 4). The resulting NC2-TATA-binding protein-DNA complex sterically hinders the recruitment of transcription factor IIB and in part of transcription factor IIA (5), and thus inhibits transcription initiation (6, 7). The NC2 complex is present on a substantial fraction of human genes (8). Besides mediating TATA-binding protein binding to TATA-containing and TATA-less promoters (9) the NC2 complex can also mobilize TATA-binding protein on the DNA (10). In addition to the well established function as transcriptional repressor (11) several studies have shown that NC2 activates transcription, in vitro and in vivo (12-16). The mechanism underlying the positive effects of NC2 on gene expression is not understood. Although the two NC2 subunits mostly function together, recent studies in S. cerevisae (17), Drosophila (18), and human provided evidence that NC2␣ and NC2␤ can associate with different proteins (19 -21). In this study, we have analyzed whether human NC2 subunits contain localization signals that permit individual crossing of the nuclear membrane or whether nuclear import of NC2␣ and NC2␤ depends on heterodimerization.During interphase the exclusive site of nucleocytoplasmic exchange is the nuclear pore complex...

The nucleoporin-like protein NLP1 (hCG1) promotes CRM1-dependent nuclear protein export

“…S1A,B). NC2b employs importin-a and/or -b and importin 13 as import-and CRM1 as export receptors, respectively (Kahle et al, 2009). Notably, under conditions of overexpression of CRM1 substrates, the cytoplasmic localization of the endogenous CRM1 cargo RanBP1 was not compromised, suggesting that the CRM1 system was not completely overloaded (supplementary material Fig.…”

“…Constructs coding for CRM1-HA (Hilliard et al, 2010), NC2b-GFP 2 (Kahle et al, 2009), NES-GFP 2 -cNLS [Rev(47-116)-GFP 2 -cNLS] and Rev68-90-GFP 2 -M9core (Hutten et al, 2008;Hutten et al, 2009) have been described previously. Human NLP1 was PCR amplified from cDNA and cloned into pMal-C2 using BamHI and HindIII, generating MBP-NLP1.…”

Oxygen sensing by Prolyl-4-Hydroxylase PHD2 within the nuclear compartment and the influence of compartimentalisation on HIF-1 signalling

“…To determine whether the identified nuclear targeting signal is sufficient to mediate nuclear import of PHD2, amino acids 180-220 of PHD2 were fused to EGFP-EGFP-GST (EEG, kindly provided by D. Doenecke). Expression of EEG in mammalian cells results in cytoplasmic localisation because it is too large to enter the nucleus by passive diffusion (Kahle et al, 2009). Fusion of PHD2 amino acids 180-220 to EEG resulted in nuclear import of the fusion protein, indicating functional NLS activity of PHD2 amino acids 180 to 220 (Fig.…”

Nuclear Transport of Wilms′ Tumour Protein Wt1 Involves Importins α and β

“…All plasmids were generated by PCR amplification using full-length cDNA of the murine Wt1 gene as template. Plasmid pEEG-C1 has been derived from pEGFP-C1 (BD Biosciences, Clontech, Heidelberg, Germany) by insertion of a BglII/XhoI GST fragment N-terminal of the multi cloning site (MCS) and a second EGFP at the N-terminus as NheI fragment [43]. Due to its molecular mass of approximately 80 kDa the EGFP-EGFP-GST fusion protein expressed from pEEG-C1 is retained in the cell cytoplasm.…”