Novel target genes of the Ah (dioxin) receptor: transcriptional induction of N-myristoyltransferase 2.

Dioxins are potent mammalian carcinogens and toxins affecting liver, skin, and immune and reproductive systems. The intracellular Ah receptor, a ligand-dependent transcription factor of the basic region/helix-loop-helix/Per-Ahr/Arnt-Sim homology domain (bHLH-PAS) protein family, mediates responses to dioxins. Target genes of the Ah receptor that mediate dioxin toxicity and carcinogenicity are, however, mostly unknown. We used 5L rat hepatoma cells to identify dioxin-inducible genes by suppression subtractive hybridization. Eleven cDNA fragments were identified that represented novel sequences or genes for which induction by dioxins had not been known. N-myristoyltransferase 2 (NMT2) is one of the later dioxin-inducible genes. Induction of NMT2 was confirmed in livers of mice in vivo. NMT2 induction was a direct consequence of Ah receptor activation in 5L cells. [(3)H]myristic acid incorporation into 5L cell proteins was inducible by dioxins, indicating that protein myristoylation is a regulated rather than a housekeeping function and that NMT activity is limiting in noninduced 5L cells. Here we show for the first time that expression of NMT2 and induced protein myristoyltransferase activity are direct responses to carcinogen exposure. Because inappropriate protein NH(2)-terminal myristoylation appears to play a role in carcinogenesis, induction of NMT2 may play a central role in dioxin carcinogenicity.

The Nsp1p carboxy-terminal domain is organized into functionally distinct coiled-coil regions required for assembly of nucleoporin subcomplexes and nucleocytoplasmic transport.

Nucleoporin Nsp1p, which has four predicted coiled-coil regions (coils 1 to 4) in the essential carboxy-terminal domain, is unique in that it is part of two distinct nuclear pore complex (NPC) subcomplexes, Nsp1p-Nup57p-Nup49p-Nic96p and Nsp1p-Nup82p-Nup159p. As shown by in vitro reconstitution, coiled-coil region 2 (residues 673 to 738) is sufficient to form heterotrimeric core complexes and can bind either Nup57p or Nup82p. Accordingly, interaction of Nup82p with Nsp1p coil 2 is competed by excess Nup57p. Strikingly, coil 3 and 4 mutants are still assembled into the core Nsp1p-Nup57p-Nup49p complex but no longer associate with Nic96p. Consistently, the Nsp1p-Nup57p-Nup49p core complex dissociates from the nuclear pores in nsp1 coil 3 and 4 mutant cells, and as a consequence, defects in nuclear protein import are observed. Finally, the nsp1-L640S temperature-sensitive mutation, which maps in coil 1, leads to a strong nuclear mRNA export defect. Thus, distinct coiled-coil regions within Nsp1p-C have separate functions that are related to the assembly of different NPC subcomplexes, nucleocytoplasmic transport, and incorporation into the nuclear pores.

Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex.

Nup116p is a GLFG nucleoporin involved in RNA export processes. We show here that Nup116p physically interacts with the Nup82p-Nsp1p-Nup159p nuclear pore subcomplex, which plays a central role in nuclear mRNA export. For this association, a sequence within the C-terminal domain of Nup116p that includes the conserved nucleoporin RNA-binding motif was sufficient and necessary. Consistent with this biochemical interaction, protein A-Nup116p and the protein A-tagged Nup116p C-terminal domain, like the members of the Nup82p complex, localized to the cytoplasmic side of the nuclear pore complex, as revealed by immunogold labeling. Finally, synthetic lethal interactions were found between mutant alleles of NUP116 and all members of the Nup82p complex. Thus, Nup116p consists of three independent functional domains: 1) the C-terminal part interacts with the Nup82p complex; 2) the Gle2p-binding sequence interacts with Gle2p/Rae1p; and 3) the GLFG domain interacts with shuttling transport receptors such as karyopherin-beta family members.