The Karyopherin Kap122p/Pdr6p Imports Both Subunits of the Transcription Factor Iia into the Nucleus

Titov, Anton A.; Blobel, Günter

doi:10.1083/jcb.147.2.235

Cited by 50 publications

(56 citation statements)

References 53 publications

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“…How each Kap recognizes specific cargo proteins has been the subject of intense study. The only characterized cargo proteins of Kap122 are two TFIIA subunits, Toa1 and Toa2 (63). In this study, we have identified four potential cargo proteins of Kap122: Wtm1, Wtm2, ␤, and ␤Ј.…”

Section: Discussionmentioning

confidence: 95%

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

Zhang

Yang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 (␣, oligomeric state unknown) and a small subunit R2 (␤ 2). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [(Fe) 2-Y⅐] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer (␤␤ ), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, ␤␤ is redistributed from the nucleus to the cytoplasm in a checkpointdependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in ␤␤ nuclear localization: the importin ␤ homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of ␤␤ nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with ␤␤ . Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of ␤␤ by Kap122 or as an anchor to retain ␤␤ in the nucleus. DNA-damage checkpoint ͉ subcellular redistributionT he levels and relative ratios of dNTP pools are important for high-fidelity DNA replication and repair (1). Failure to increase dNTP levels at the G 1 -to-S transition of the cell cycle is a lethal event at cellular level (2, 3). Conversely, elevated dNTP pools throughout the cell cycle lead to increased mutation rates (4-6). Imbalance in dNTP pools also contributes to mutagenesis by reducing the fidelity of DNA polymerases (7-9). Eukaryotic cells have evolved complex surveillance mechanisms (i.e., checkpoints) to ensure proper dNTP pool sizes during the normal cell-cycle progression and in response to genotoxic stress (3, 10-14). A major target of such checkpoint regulation is ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleoside diphosphate to deoxyribonucleoside diphosphate, an essential step in de novo biosynthesis of dNTPs (15).Class I RNRs were identified originally in Escherichia coli and are conserved from yeast to mammal (16). The mechanisms of enzymatic catalysis (17) and allosteric regulation (18, 19) have been studied extensively in E. coli and, more recently, in mice (20, 21). The archetype RNR holoenzyme consists of a large subunit R1 (␣, whose oligomeric state in eukaryotes is not completely understood) (22) and a homodimeric small subunit (␤ 2 ) (20). The eukaryotic R1 contains the catalytic site, an effector site that controls substrate specificity, an activity site that controls turnover, and a weak ATP-binding site that cont...

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Section: Discussionmentioning

confidence: 95%

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

Zhang

Yang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Hence, despite their functional divergence these two histone-fold heterodimers rely on the same import mechanism to translocate through the NPC. Interestingly, for importin 13 the most similar protein from S. cerevisiae is Pdr6p/karyopherin 122, which translocates the NC2 antagonist transcription factor IIA into the nucleus (68). The identity of Pdr6p with importin 13 is too low to regard it as a clear orthologue (40).…”

Section: Nc2␣ (mentioning

confidence: 99%

Regulation of Nuclear Import and Export of Negative Cofactor 2

Kahle

Piaia

Neimanis

et al. 2009

Journal of Biological Chemistry

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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...

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“…The localization of GR-GFP was monitored in 14 yeast strains each deficient in a different nuclear transport receptor: CRMI (Stade et al, 1997), CSEI (Xiao et al, 1993), Kap95 (Iovine et al, 1995), Kap104 (Aitchison et al, 1996), Kap123 (Seedorf and Silver, 1997), LOS1 (Hopper et al, 1980), MSN5 (Kaffman et al, 1998a), MTR10 (Kadowaki et al, 1994), NMD5 (He and Jacobson, 1995), PSEI (Seedorf and Silver, 1997), SRP1 (Yano et al, 1992), SXM1 (Seedorf and Silver, 1997), Kap114 (Morehouse et al, 1999;Pemberton et al, 1999), and PDR6 (Titov and Blobel, 1999). Yeast strains PSY580, pse1-1, PSY1200, and PSY902 have been described (Seedorf and Silver, 1997).…”

Section: Yeast Strains Handling Transformations and Localization Smentioning

confidence: 99%

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

Freedman

2004

MBoC

192

151

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The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin ␣ selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin ␣-importin ␤ heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin ␣ bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding. INTRODUCTIONTo survive in a complex environment, cells sense external cues and commonly respond by altering gene expression, in part by regulating the intracellular localization and activity of transcriptional regulatory factors. For example, the intracellular localization of the yeast Pho4 and mammalian SREBP factors is altered in response to changes in external phosphate and circulating sterol concentrations, respectively (Kaffman et al., 1998b;Nagoshi et al., 1999). Similarly, changes in blood glucose levels and a wide range of physiological stress signals modulate the circulating level of glucocorticoids, changing the activity and localization of the glucocorticoid receptor (GR). Within minutes of glucocorticoid binding, GR enters the nucleus and activates or represses target gene transcription (Picard and Yamamoto, 1987). However, hormone binding is reversible; after hormone withdrawal, GR locates back to the cytoplasm (t 1/2 ϭ 10 h; Hache et al., 1999).Proteins and RNAs enter and exit the nucleus through the nuclear pore, a 125-MDa complex in the nuclear envelope (Stoffler et al., 1999). Small molecules readily diffuse through the nuclear pore, whereas molecules greater than ϳ40 kDa require import and export machinery for transport (Davis, 1995;Pante and Aebi, 1995). The first nuclear localization sequence (NLS) was identified in the SV40 large T-antigen (Kalderon et al., 1984). Development of an in vitro nuclear import assay facilitated the identification of two proteins that import substrates containing SV40 NLS-like domains. The adapter molecule importin ␣ binds to the NLS of the substrate and the importin ␤ transport receptor, creating a trimeric complex that imports the substrate into the nucleus through the nuclear pore (Strom and Weis, 2001).Based on similarity to importin ␤, a family of nuclear import and export receptors was identified and shown to transport a wide variety of proteins and RNAs into and out of the nucleus. After reaching the nucleoplasm, import receptors bind the...

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The Karyopherin Kap122p/Pdr6p Imports Both Subunits of the Transcription Factor Iia into the Nucleus

Cited by 50 publications

References 53 publications

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

Regulation of Nuclear Import and Export of Negative Cofactor 2

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

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