[Abstract unavailable

SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the ⌿KXE motif, where ⌿ represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IB␣, containing the ⌿KXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivo requires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation. SUMO-11 is a small ubiquitin-related modifier (also known as sentrin, GMP1, UBL1, PIC1, or SMT3 in yeast) that has been found covalently conjugated to various cellular proteins (for reviews see Refs. 1-3). Several substrates for SUMO-1 have been reported: the RanGTPase-activating protein (Ran-GAP1) (4, 5) and Ran-binding protein 2 (6) implicated in nucleocytoplasmic trafficking; the promyelocytic leukemia protein (PML) and Sp100 (7) found in subnuclear structures known as PML oncogenic domains or PODs; the IB␣ inhibitor of the transcription factor nuclear factor B, implicated in the control of immune and inflammatory responses (8); and the tumor suppressor protein p53 (9, 10). Consequently, "SUMOylation" plays a role in multiple vital cellular processes such as oncogenesis, cell cycle control, apoptosis, and response to virus infection.SUMO-1 is conjugated to a target protein by a pathway that is distinct from but analogous to ubiquitin conjugation. Like ubiquitin, SUMO-1 is proteolytically processed to expose its mature C terminus by recently described SUMO-1-specific proteases variously called Ulp1 and Ulp2 in yeast (11,12) or SENP1 and SUSP-1 in vertebrates (13-15). Ulp1, Ulp2, and SENP1, but not SUSP-1, are capable of both deconjugating SUMO-1 from modified proteins and removing four amino acids from the C terminus of the 101-amino acid SUMO-1 precursor to generate the mature 97-amino acid form. SUMO-1 addition is accomplished by a thioester cascade, with SUMO-1 first being activated by a heterodimeric SUMO-1-activating enzyme (SAE) that adenylates the C-terminal glycine of before catalyzing the formation of a thioester bond between the C terminus of SUMO-1 and a cysteine residue in SAE. In a transesterification reaction SUMO-1 is transferred from the SAE to the SUMO-1-conjugating enzyme Ubc9, which catalyzes the formation of an isopeptide bond between the C terminus of SUMO-1 and the ⑀-amino group of a lysine residue of the target protein (6, 20 -23). Ubc9 is required for cell cycle progression in yeast (24). Unlike ubiquitin conjugation, SUMO-1 modification of target proteins in vitro is not dependent on the equivalent of an E3 protein ligase (17,19).Here, we demonstrate that a short sequence containing the consensus ⌿KXE, where ⌿ represents a large hydrophobic amino acid, co...

Chromosome maintenance region 1 (CRM1), a protein that shares sequence similarities with the karyopherin beta family of proteins involved in nuclear import pathway, was shown to form a complex with the leucine-rich nuclear export signal (NES). This interaction was inhibited by leptomycin B, a drug that prevents the function of the CRM1 protein in yeast. To analyze the role of the CRM1-NES interaction in nuclear export, a transport assay based on semipermeabilized cells was developed. In this system, which reconstituted NES-, cytosol-, and energy-dependent nuclear export, leptomycin B specifically blocked export of NES-containing proteins. Thus, the CRM1 protein could act as a NES receptor involved in nuclear protein export.

Interferons (IFN) α/β and γ induce the formation of two transcriptional activators: gamma-activating factor (GAF) and interferon-stimulated gamma factor 3 (ISGF3). We report a natural heterozygous germline STAT1 mutation associated with susceptibility to mycobacterial but not viral disease. This mutation causes a loss of GAF and ISGF3 activation but is dominant for one cellular phenotype and recessive for the other. It impairs the nuclear accumulation of GAF but not of ISGF3 in heterozygous cells stimulated by IFNs. Thus, the antimycobacterial, but not the antiviral, effects of human IFNs are principally mediated by GAF.

No abstract

The insertion of a DNA copy of its RNA genome into a chromosome of the host cell is mediated by the viral integrase with the help of mostly uncharacterized cellular cofactors. We have recently described that the transcriptional co-activator LEDGF/p75 strongly interacts with HIV-1 integrase. Here we show that interaction of HIV-1 integrase with LEDGF/p75 is important for viral replication. Using multiple approaches including two-hybrid interaction studies, random and directed mutagenesis, we could demonstrate that HIV-1 virus harboring a single mutation that disrupts integrase-LEDGF/p75 interaction, resulted in defective HIV-1 replication. Furthermore, we found that LEDGF/p75 tethers HIV-1 integrase to chromosomes and that this interaction may be important for the integration process and the replication of HIV-1.

Transcriptional activation of nuclear factor B (NF-B) is mediated by signal-induced phosphorylation and degradation of its inhibitor, IB␣. However, NF-B activation induces rapid resynthesis of IB␣, which is responsible for post-induction repression of transcription. Newly synthesized IB␣ translocates to the nucleus, where it dissociates NF-B from DNA and transports NF-B from the nucleus to the cytoplasm in a nuclear export sequence-dependent process that is sensitive to leptomycin B (LMB). In the present study, LMB was used as a tool to inhibit nuclear export sequence-mediated nuclear protein export and evaluate the consequences for regulation of NF-B-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-B-dependent transcriptional activation mediated by interleukin 1␤ or tumor necrosis factor ␣. This is a consequence of the inhibition of signal-induced degradation of IB␣. Although LMB treatment does not affect the signal transduction pathway leading to IB␣ degradation, it blocks IB␣ nuclear export. IB␣ is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IB␣ is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IB␣ is therefore essential for maintaining a low level of IB␣ in the nucleus and allowing NF-B to be transcriptionally active upon cell stimulation.The NF-B 1 /Rel family of transcription factors is implicated in regulation of the expression of a number of cellular genes involved in immune responses, inflammation ,and apoptosis (for recent reviews, see Refs. 1-3). In vertebrates, the NF-B family of proteins is composed of transcriptionally active p65/ Rel A (4, 5), c-Rel (6), or Rel B (7) and transcriptionally silent p50/NF-B1 (8, 9) or p52/NF-B2 (10 -12). All NF-B proteins share a conserved region known as the Rel homology domain that contains the nuclear localization signal as well as the dimerization and DNA binding functions. The NF-B form activated by extracellular signals is composed of p50 and p65. NF-B transcriptional activity is controlled by inhibitor IB proteins that contain ankyrin repeat domains. Association of p50/p65 with IB not only occludes the nuclear localization sequence of p50 and p65, leading to cytoplasmic sequestration, but also prevents NF-B DNA binding activity. Several IBs have been described including IB␣ (13), IB␤ (14), IB⑀ (15), and Bcl-3 (16). Additionally, the precursors of p50 (p105) and p52 (p100) possess inhibitory ankyrin repeat domains that in isolation are known as IB␥ (17-19) and IB␦ (20, 21), respectively.IB␣ is organized in three domains: (a) an unstructured amino-terminal (aa 1-72) signal response domain, (b) a central region (aa 73-242) consisting of five ankyrin repeat domains, and (c) a carboxyl-terminal region (aa 243-317) containing a highly acidic domain (aa 276 -317) that is bound to the ankyrin repeat domain by a protease-sensitive linker (aa 243-275) and is protected by bound p65 (22)....

The evolutionarily conserved mRNA export receptor Mex67/NXF1 associates with mRNAs through its adaptor, Yra1/REF, allowing mRNA ribonucleoprotein (mRNP) exit through nuclear pores. However, alternate adaptors should exist, since Yra1 is dispensable for mRNA export in Drosophila and Caenorhabditis elegans. Here we report that Mex67 interacts directly with Nab2, an essential shuttling mRNA-binding protein required for export. We further show that Yra1 enhances the interaction between Nab2 and Mex67, and becomes dispensable in cells overexpressing Nab2 or Mex67. These observations appoint Nab2 as a potential adaptor for Mex67, and define Yra1/REF as a cofactor stabilizing the adaptor-receptor interaction. Importantly, Yra1 ubiquitination by the E3 ligase Tom1 promotes its dissociation from mRNP before export. Finally, loss of perinuclear Mlp proteins suppresses the growth defects of Tom1 and Yra1 ubiquitination mutants, suggesting that Tom1-mediated dissociation of Yra1 from Nab2-bound mRNAs is part of a surveillance mechanism at the pore, ensuring export of mature mRNPs only.[Keywords: Mex67/NXF1; Nab2; Yra1/REF; Tom1; mRNA export; ubiquitination] Supplemental material is available at http://www.genesdev.org.