Nuclear Localization Signal Receptor Affinity Correlates with in Vivo Localization in Saccharomyces cerevisiae

Hodel, A.E.; Harreman, Michelle T.; Pulliam, Kanika F.; Harben, Mary Elizabeth; Holmes, Jordan S.; Hodel, Mary R.; Berland, Keith M.; Corbett, Anita H.

doi:10.1074/jbc.m601718200

Cited by 99 publications

(124 citation statements)

References 53 publications

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“…The functional significance of altered NLS-binding affinity for importin-α and its effect on localisation of NLS peptides has been reported in yeast cells (Hodel et al, 2006). Here, we show that the binding affinity for importin-α also influences nuclear import of whole proteins in mammalian cells.…”

Section: Structure Of the Binding Interface In The Importin-α-ar Complexsupporting

confidence: 63%

Structural basis for the nuclear import of the human androgen receptor

Cutress

Whitaker

Mills

et al. 2008

Journal of Cell Science

197

162

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Ligand-dependent nuclear import is crucial for the function of the androgen receptor (AR) in both health and disease. The unliganded AR is retained in the cytoplasm but, on binding 5α-dihydrotestosterone, it translocates into the nucleus and alters transcription of its target genes. Nuclear import of AR is mediated by the nuclear import factor importin-α, which functions as a receptor that recognises and binds to specific nuclear localisation signal (NLS) motifs on cargo proteins. We show here that the AR binds to importin-α directly, albeit more weakly than the NLS of SV40 or nucleoplasmin. We describe the 2.6-Å-resolution crystal structure of the importin-α–AR-NLS complex, and show that the AR binds to the major NLS-binding site on importin-α in a manner different from most other NLSs. Finally, we have shown that pathological mutations within the NLS of AR that are associated with prostate cancer and androgen-insensitivity syndrome reduce the binding affinity to importin-α and, subsequently, retard nuclear import; surprisingly, however, the transcriptional activity of these mutants varies widely. Thus, in addition to its function in the nuclear import of AR, the NLS in the hinge region of AR has a separate, quite distinct role on transactivation, which becomes apparent once nuclear import has been achieved.

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Section: Structure Of the Binding Interface In The Importin-α-ar Complexsupporting

confidence: 63%

Structural basis for the nuclear import of the human androgen receptor

Cutress

Whitaker

Mills

et al. 2008

Journal of Cell Science

197

162

View full text Add to dashboard Cite

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“…NLS-GFP import assay: The NLS-GFP import assay was performed as described previously (Shulga et al 1996;Hodel et al 2006). Cells were resuspended in glucose-containing synthetic media pre-equilibrated to 25°(permissive), 37°(srp1-31, srp1-E402Q), or 18°(srp1-55).…”

Section: Methodsmentioning

confidence: 99%

“…cNLS cargo recognition and 1 transport is mediated by a soluble heterodimeric protein receptor composed of an adapter, importin/ karyopherin-a, which recognizes the cNLS cargo in the cytoplasm and a carrier, importin/karyopherin-b, which targets the complex to the nuclear pore complex (NPC) for transport (Gö rlich et al 1995;Bayliss et al 2000;Liu and Stewart 2005). Significant evidence has accumulated to support the idea that rates of import into the nucleus are largely determined by interaction between the NLS cargo and the NLS receptor (Hodel et al 2006;Timney et al 2006;Riddick and Macara 2007), making recognition of the NLS cargo by the NLS receptor essentially the rate-limiting step in the process of nuclear protein import.…”

mentioning

confidence: 99%

The Classical Nuclear Localization Signal Receptor, Importin-α, Is Required for Efficient Transition Through the G1/S Stage of the Cell Cycle inSaccharomyces cerevisiae

et al. 2009

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There is significant evidence linking nucleocytoplasmic transport to cell cycle control. The budding yeast, Saccharomyces cerevisiae, serves as an ideal model system for studying transport events critical to cell cycle progression because the nuclear envelope remains intact throughout the cell cycle. Previous studies linked the classical nuclear localization signal (cNLS) receptor, importin-a/Srp1, to the G 2 /M transition of the cell cycle. Here, we utilize two engineered mutants of importin-a/Srp1 with specific molecular defects to explore how protein import affects cell cycle progression. One mutant, Srp1-E402Q, is defective in binding to cNLS cargoes that contain two clusters of basic residues termed a bipartite cNLS. The other mutant, Srp1-55, has defects in release of cNLS cargoes into the nucleus. Consistent with distinct in vivo functional consequences for each of the Srp1 mutants analyzed, we find that overexpression of different nuclear transport factors can suppress the temperature-sensitive growth defects of each mutant. Studies aimed at understanding how each of these mutants affects cell cycle progression reveal a profound defect at the G 1 to S phase transition in both srp1-E402Q and srp1-55 mutants as well as a modest G 1 /S defect in the temperature-sensitive srp1-31 mutant, which was previously implicated in G 2 /M. We take advantage of the characterized defects in the srp1-E402Q and srp1-55 mutants to predict candidate cargo proteins likely to be affected in these mutants and provide evidence that three of these cargoes, Cdc45, Yox1, and Mcm10, are not efficiently localized to the nucleus in importin-a mutants. These results reveal that the classical nuclear protein import pathway makes important contributions to the G 1 /S cell cycle transition.

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“…Increased binding affinity for importins has been shown to increase the efficiency of nuclear import (Hodel et al, 2006;Jans et al, 2000), and we reasoned that if a complex of PB1 and PA had greater affinity for RanBP5 than PB1 alone this would explain the requirement of PA for the efficient nuclear import of PB1. However, the ability of PB1-TAP to co-purify RanBP5 was not increased by PA (Fig.…”

Section: Ranbp5 Binding Efficiency Of Pb1 Affects Nuclear Localizationmentioning

confidence: 99%

Characterization of the interaction between the influenza A virus polymerase subunit PB1 and the host nuclear import factor Ran-binding protein 5

Hutchinson

Orr

Liu

et al. 2011

Journal of General Virology

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The influenza A virus RNA polymerase is a heterotrimer that transcribes and replicates the viral genome in the cell nucleus. Newly synthesized RNA polymerase subunits must therefore be imported into the nucleus during an infection. While various models have been proposed for this process, the consensus is that the polymerase basic protein PB1 and polymerase acidic protein PA subunits form a dimer in the cytoplasm and are transported into the nucleus by the betaimportin Ran-binding protein 5 (RanBP5), with the PB2 subunit imported separately to complete the trimeric complex. In this study, we characterized the interaction of PB1 with RanBP5 further and assessed its importance for viral growth. In particular, we found that the N-terminal region of PB1 mediates its binding to RanBP5 and that basic residues in a nuclear localization signal are required for RanBP5 binding. Mutating these basic residues to alanines does not prevent PB1 forming a dimer with PA, but does reduce RanBP5 binding. RanBP5-binding mutations reduce, though do not entirely prevent, the nuclear accumulation of PB1. Furthermore, mutations affecting RanBP5 binding are incompatible with or severely attenuate viral growth, providing further support for a key role for RanBP5 in the influenza A virus life cycle. INTRODUCTIONInfluenza A virus is the prototypic orthomyxovirus and a serious pathogen of humans and animals. The viral genome consists of eight segments of negative-sense, ssRNA, which are encapsidated as viral ribonucleoprotein complexes (vRNPs) by multiple copies of nucleoprotein (NP) and the trimeric RNA-dependent RNA polymerase (RdRP; consisting of the polymerase basic proteins PB1 and PB2, and the polymerase acidic protein PA) (Palese & Shaw, 2007). Unusually among RNA viruses, orthomyxoviruses replicate their genomes in the nucleus. Consequently, newly synthesized viral proteins must be imported into the nucleus to allow the assembly and export of new vRNPs, as well as to regulate host processes in the infected cell. Of the proteins expressed by influenza A virus, nuclear import is observed for the three polymerase proteins and NP, as well as matrix (M1) protein, non-structural (NS1) protein, nuclear export protein (NEP) and PB1-F2 (Chen et al., 2001;Smith et al., 1987). Nuclear import of structures larger than 20-30 kDa is selective and energy dependent, requiring binding of nuclear import factors (NIFs) through nuclear localization signals (NLSs) on import substrates (Görlich & Kutay, 1999). A number of influenza A virus proteins have been shown to contain NLSs and to bind NIFs Naito et al., 2007; Resa-Infante et al., 2008;Tarendeau et al., 2007). PB1 and PA, despite both having signals that can promote nuclear import when individually expressed (Akkina et al., 1987;Nath & Nayak, 1990;Nieto et al., 1994), do not accumulate efficiently in the nucleus unless expressed together, a requirement that appears to be particularly marked for PB1 (Fodor & Smith, 2004;Huet et al., 2010;Nieto et al., 1992). Although all possible pairwise interact...

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Nuclear Localization Signal Receptor Affinity Correlates with in Vivo Localization in Saccharomyces cerevisiae

Cited by 99 publications

References 53 publications

Structural basis for the nuclear import of the human androgen receptor

Structural basis for the nuclear import of the human androgen receptor

The Classical Nuclear Localization Signal Receptor, Importin-α, Is Required for Efficient Transition Through the G1/S Stage of the Cell Cycle inSaccharomyces cerevisiae

Characterization of the interaction between the influenza A virus polymerase subunit PB1 and the host nuclear import factor Ran-binding protein 5

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