The Nuclear Pore Complex

Antizyme-1 (AZ1) is a protein that negatively regulates polyamine synthesis by inhibiting the key synthetic enzyme ornithine decarboxylase and targeting it for degradation by the 26 S proteasome. Recent work shows that antizyme protein translocates to the nucleus during mouse development (Gritli-Linde, A., Nilssom, J., Bohlooly, Y. M., Heby, O., and Linde, A. (2001) Dev. Dyn. 220, 259 -275). However, the significance and mechanism of this phenomenon remain unclear. In this study, we expressed AZ1 fused with enhanced green fluorescent protein (EGFP) to study its localization in a living cell. We found that EGFP-AZ1 was predominantly localized in the cytoplasm and that treatment with leptomycin B, a specific inhibitor of chromosomal region maintenance 1 (CRM1) induced nuclear accumulation of EGFP-AZ1 in Chinese hamster ovary and NIH3T3 cells. Two independent nuclear export signal (NES) sequences, each containing essential hydrophobic residues, were identified in the 50 N-terminal amino acid residues and in the central part of AZ1. The activity of the second NES was inhibited by an N-terminal adjacent region and was only revealed in N-terminal truncated constructs. Both NESs were active when fused to an artificial nuclear protein SV40-NLS-EGFP-EGFP. The ability of AZ1 to shuttle between the nucleus and the cytoplasm suggests that it has a novel function in the nucleus.Polyamines are ubiquitous organic cations with tightly regulated cellular concentrations and are essential for cell growth and development (1, 2). Antizyme (AZ) 1 was first described as an inhibitor of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis (3). When cellular polyamine levels rise, AZ is induced and then binds to the monomeric form of ODC (4, 5), which makes the enzyme a preferential substrate for proteolysis by the 26 S proteasome without ubiquitination (6). AZ also inhibits the uptake of extracellular polyamines (7,8). There are at least three independent antizyme isoforms conserved among mammals. AZ1 and AZ2 have a wide tissue distribution, whereas AZ3 is testis-specific (9, 10). Expression of AZs is induced by a unique translational frameshift mechanism (11). For AZ1, when cellular polyamine levels are low, the polypeptide corresponding to amino acid residues 1-68 is produced and translation is terminated at the following UGA codon (11). When cellular polyamine levels are high, ϩ1 frameshifting occurs after the 68th codon is decoded, resulting in the full-length antizyme protein (amino acid residues 1-227). All of the known regulatory activities of AZ1 are present in amino acid residues 69 -227 (12). A second AUG exists in AZ1 mRNA at the 34th codon, and this second AUG is also utilized as initiation codon (11, 13). The two resulting translation products have different half-lives (14). Furthermore, it has been suggested that the N-terminal region of the translation product from the first AUG contains a putative mitochondrial targeting signal and thus the distribution of AZ1 may depend on this region (15). It is curre...

Section: Fig 7 Mutational Analysis Of Nes2mentioning

confidence: 99%

Identification of Nuclear Export Signals in Antizyme-1

Murai

Murakami

Matsufuji

2003

“…The assembled structures, termed nuclear pore complexes, are composed of subunits of nucleoporins, and act as the sole gatekeepers controlling the exchange of materials between the two compartments (9,10). Although nuclear pore complexes are freely permeable to small molecules (such as water and ions) with passive diffusion, they restrict the movement of larger molecules (such as proteins and RNAs) across the nuclear envelope.…”

mentioning

confidence: 99%

Importin 4 Is Responsible for Ligand-independent Nuclear Translocation of Vitamin D Receptor

Miyauchi

Michigami

Sakaguchi

et al. 2005

Vitamin D receptor (VDR) is localized in nuclei and acts as a ligand-dependent transcription factor. To clarify the molecular mechanisms underlying the nuclear translocation of VDR, we utilized an in vitro nuclear transport assay using digitonin-permeabilized semi-intact cells. In this assay, recombinant whole VDR-(4 -427) and a truncated mutant VDR-(4 -232) lacking the carboxyl terminus of VDR were imported to nuclei even in the absence of ligand. In contrast, VDR-(91-427) lacking the amino-terminal DNA-binding domain was not imported to nuclei in the absence of ligand, and was efficiently imported in its liganded form. These results suggested that there are two distinct mechanisms underlying the nuclear transport of VDR; ligand-dependent and -independent pathways, and that the different regions of VDR are responsible for these processes. Therefore, we performed the yeast two-hybrid screening using VDR-(4 -232) as the bait to explore the molecules responsible for ligand-independent nuclear translocation of VDR, and have identified importin 4 as an interacting protein. In the reconstruction experiments where transport factors were applied as recombinant proteins, recombinant importin 4 facilitated nuclear translocation of VDR regardless of its ligand, whereas importin ␤ failed in transporting VDR even in the presence of ligand. In conclusion, importin 4, not importin ␤, is responsible for the ligandindependent nuclear translocation of VDR.The active form of vitamin D, 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 D 3 ) 3 regulates various biological events through its direct actions on gene expression in its target organs such as intestine, kidney, and bone (1, 2). Its cognate receptor, vitamin D receptor (VDR), belongs to the nuclear receptor superfamily, and plays crucial roles in the physiological actions of 1␣,25(OH) 2 D 3 , including calcium homeostasis and bone metabolism (1, 2). Nuclear receptors including VDR act as ligandinducible transcription factors, and consist of several functional domains to regulate the expression of the target genes (1-3). Utilizing its DNA-binding domain, VDR recognizes a vitamin D-responsive element (VDRE) in the target genes, and regulates the expressions via forming complexes with retinoid X receptor (RXR) and transcriptional co-factors on its ligand-binding domain. To fulfill these events on the genome, nuclear receptors must be localized in nuclei. However, it seems that the subcellular distributions of the nuclear receptors do not resemble each other, unlike their transcriptional regulatory systems. Several reports have demonstrated that VDR is located in nuclei even in the absence of 1␣,25(OH) 2 D 3 and is accumulated in nuclei to a greater extent by addition of the ligand (4 -6). These observations are in contrast with the cases of other nuclear receptors, glucocorticoid receptor or estrogen receptor; the former translocates from the cytoplasm into nuclei only in the presence of the ligand, and the latter is located in nuclei regardless of the ligand (7, 8). The obvious diffe...

“…All nucleocytoplasmic transport of macromolecules takes place through the NPC (17). The NPC consists of a large number of proteins termed nucleoporins (18).…”

mentioning

confidence: 99%

The Yeast Nucleoporin Nup2p Is Involved in Nuclear Export of Importin α/Srp1p

Booth

Belanger

Sannella

et al. 1999

Self Cite

The importin ␣⅐␤ heterodimer mediates nuclear import of proteins containing classical nuclear localization signals. After carrying its cargo into the nucleus, the importin dimer dissociates, and Srp1p (the yeast importin ␣ subunit) is recycled to the cytoplasm in a complex with Cse1p and RanGTP. Nup2p is a yeast FXFG nucleoporin that contains a Ran-binding domain. We find that export of Srp1p from the nucleus is impaired in ⌬nup2 mutants. Also, Srp1p fusion proteins accumulate at the nuclear rim in wild-type cells but accumulate in the nuclear interior in ⌬nup2 cells. A deletion of NUP2 shows genetic interactions with mutants in SRP1 and PRP20, which encodes the Ran nucleotide exchange factor. Srp1p binds directly to an Nterminal domain of Nup2p. This region of Nup2p is sufficient to allow accumulation of an Srp1p fusion protein at the nuclear rim, but the C-terminal Ran-binding domain of Nup2p is required for efficient Srp1p export.