Antagonistic effects of NES and NLS motifs determine S. cerevisiae Rna1p subcellular distribution

1999 ◽  
Vol 112 (3) ◽  
pp. 339-347 ◽  
Author(s):  
W. Feng ◽  
A.L. Benko ◽  
J.H. Lee ◽  
D.R. Stanford ◽  
A.K. Hopper
Keyword(s):  
Nuclear Membrane ◽  
Nuclear Export ◽  
Nuclear Localization Sequence ◽  
Gtpase Activating Protein ◽  
Gtp Hydrolysis ◽  
Nuclear Interior ◽  
Localization Sequence ◽  
Nuclear Location ◽  
Ran Gtp ◽  
Gtpase Ran

Nucleus/cytosol exchange requires a GTPase, Ran. In yeast Rna1p is the GTPase activating protein for Ran (RanGAP) and Prp20p is the Ran GDP/GTP exchange factor (GEF). RanGAP is primarily cytosolic and GEF is nuclear. Their subcellular distributions led to the prediction that Ran-GTP hydrolysis takes place solely in the cytosol and GDP/GTP exchange solely in the nucleus. Current models propose that the Ran-GTP/Ran-GDP gradient across the nuclear membrane determines the direction of exchange. We provide three lines of evidence that Rna1p enters and leaves the nuclear interior. (1) Rna1p possesses leucine-rich nuclear export sequences (NES) that are able to relocate a passenger karyophilic protein to the cytosol; alterations of consensus residues re-establish nuclear location. (2) Rna1p possesses other sequences that function as a novel nuclear localization sequence able to deliver a passenger cytosolic protein to the nucleus. (3) Endogenous Rna1p location is dependent upon Xpo1p/Crm1p, the yeast exportin for leucine-rich NES-containing proteins. The data support the hypothesis that Rna1p exists on both sides of the nuclear membrane, perhaps regulating the Ran-GTP/Ran-GDP gradient, participating in a complete RanGTPase nuclear cycle or serving a novel function.

scholarly journals Role of Nuclear Pools of Aminoacyl-tRNA Synthetases in tRNA Nuclear Export

2001 ◽  
Vol 12 (5) ◽  
pp. 1381-1392 ◽  
Author(s):  
Abul K. Azad ◽  
David R. Stanford ◽  
Srimonti Sarkar ◽  
Anita K. Hopper
Keyword(s):  
Nuclear Localization ◽  
Nuclear Export ◽  
Trna Synthetase ◽  
Nuclear Localization Sequence ◽  
Sequence Alignments ◽  
Trna Aminoacylation ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Localization Sequence ◽  
Nuclear Location

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export ( Lund and Dahlberg, 1998 ; Sarkar et al., 1999 ; Grosshans et al., 2000a ) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.

scholarly journals Regulation of mRNA export through API5 and nuclear FGF2 interaction

2020 ◽  
Vol 48 (11) ◽  
pp. 6340-6352 ◽  
Author(s):  
Seoung Min Bong ◽  
Seung-Hyun Bae ◽  
Bomin Song ◽  
HyeRan Gwak ◽  
Seung-Won Yang ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Export ◽  
Mrna Export ◽  
Structural Basis ◽  
Nuclear Localization Sequence ◽  
Physical Interaction ◽  
Dynamic Regulation ◽  
Localization Sequence ◽  
Localization Signal ◽  
Apoptosis Inhibitor

Abstract API5 (APoptosis Inhibitor 5) and nuclear FGF2 (Fibroblast Growth Factor 2) are upregulated in various human cancers and are correlated with poor prognosis. Although their physical interaction has been identified, the function related to the resulting complex is unknown. Here, we determined the crystal structure of the API5–FGF2 complex and identified critical residues driving the protein interaction. These findings provided a structural basis for the nuclear localization of the FGF2 isoform lacking a canonical nuclear localization signal and identified a cryptic nuclear localization sequence in FGF2. The interaction between API5 and FGF2 was important for mRNA nuclear export through both the TREX and eIF4E/LRPPRC mRNA export complexes, thus regulating the export of bulk mRNA and specific mRNAs containing eIF4E sensitivity elements, such as c-MYC and cyclin D1. These data show the newly identified molecular function of API5 and nuclear FGF2, and provide a clue to understanding the dynamic regulation of mRNA export.

scholarly journals Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

1998 ◽  
Vol 143 (7) ◽  
pp. 1801-1812 ◽  
Author(s):  
Peter Bangs ◽  
Brian Burke ◽  
Christine Powers ◽  
Roger Craig ◽  
Aruna Purohit ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Ectopic Expression ◽  
Coiled Coil ◽  
Full Length ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Nuclear Interior ◽  
Mammalian Cell Lines ◽  
Localization Sequence

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

scholarly journals The checkpoint-dependent nuclear accumulation of Rho1p exchange factor Rgf1p is important for tolerance to chronic replication stress

2014 ◽  
Vol 25 (7) ◽  
pp. 1137-1150 ◽  
Author(s):  
Sofía Muñoz ◽  
Elvira Manjón ◽  
Patricia García ◽  
Per Sunnerhagen ◽  
Yolanda Sánchez
Keyword(s):  
Rho Gtpases ◽  
Nuclear Export ◽  
Replication Stress ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
General Interest ◽  
Nucleotide Exchange ◽  
Replication Checkpoint ◽  
Exchange Factor ◽  
Localization Sequence

Guanine nucleotide exchange factors control many aspects of cell morphogenesis by turning on Rho-GTPases. The fission yeast exchange factor Rgf1p (Rho gef1) specifically regulates Rho1p during polarized growth and localizes to cortical sites. Here we report that Rgf1p is relocalized to the cell nucleus during the stalled replication caused by hydroxyurea (HU). Import to the nucleus is mediated by a nuclear localization sequence at the N-terminus of Rgf1p, whereas release into the cytoplasm requires two leucine-rich nuclear export sequences at the C-terminus. Moreover, Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and the DNA replication checkpoint kinase Cds1p. Both proteins control the nuclear accumulation of Rgf1p by inhibition of its nuclear export. A mutant, Rgf1p-9A, that substitutes nine serine potential phosphorylation Cds1p sites for alanine fails to accumulate in the nucleus in response to replication stress, and this correlates with a severe defect in survival in the presence of HU. In conclusion, we propose that the regulation of Rgf1p could be part of the mechanism by which Cds1p and Rad24p promote survival in the presence of chronic replication stress. It will be of general interest to understand whether the same is true for homologues of Rgf1p in budding yeast and higher eukaryotes.

Fragile X family members have important and non-overlapping functions

2011 ◽  
Vol 2 (5) ◽  
pp. 343-352 ◽  
Author(s):  
Claudia Winograd ◽  
Stephanie Ceman
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Family Members ◽  
Nuclear Localization Sequence ◽  
Interaction Domain ◽  
Protein Protein Interaction ◽  
Localization Sequence ◽  
Protein Functional Domains

AbstractThe fragile X family of genes encodes a small family of RNA binding proteins including FMRP, FXR1P and FXR2P that were identified in the 1990s. All three members are encoded by 17 exons and show alternative splicing at the 3′ ends of their respective transcripts. They share significant homology in the protein functional domains, including the Tudor domains, the nuclear localization sequence, a protein-protein interaction domain, the KH1 and KH2 domains and the nuclear export sequence. Fragile X family members are found throughout the animal kingdom, although all three members are not consistently present in species outside of mammals: only two family members are present in the avian species examined, Gallus gallus and Taeniopygia guttata, and in the frog Xenopus tropicalis. Although present in many tissues, the functions of the fragile X family members differ, which are particularly evident in knockout studies performed in animals. The fragile X family members play roles in normal neuronal function and in the case of FXR1, in muscle function.

Relationship between the function and the location of G1 cyclins inS. cerevisiae

2001 ◽  
Vol 114 (24) ◽  
pp. 4599-4611 ◽  
Author(s):  
Nicholas P. Edgington ◽  
Bruce Futcher
Keyword(s):  
Nuclear Localization ◽  
Cellular Localization ◽  
Subcellular Location ◽  
Nuclear Localization Sequence ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinases ◽  
G1 Cyclins ◽  
C Terminus ◽  
Localization Sequence ◽  
Nuclear Location

The Saccharomyces cerevisiae cyclin-dependent kinase Cdc28 forms complexes with nine different cyclins to promote cell division. These nine cyclin-Cdc28 complexes have different roles, but share the same catalytic subunit; thus, it is not clear how substrate specificity is achieved. One possible mechanism is specific sub-cellular localization of specific complexes. We investigated the location of two G1 cyclins using fractionation and microscopy. In addition, we developed ‘forced localization’ cassettes, which direct proteins to particular locations, to test the importance of localization. Cln2 was found in both nucleus and cytoplasm. A substrate of Cln2, Sic1, was also in both compartments. Cytoplasmic Cln2 was concentrated at sites of polarized growth. Forced localization showed that some functions of Cln2 required a cytoplasmic location, while other functions required a nuclear location. In addition, one function apparently required shuttling between the two compartments. The G1 cyclin Cln3 required nuclear localization. An autonomous, nuclear localization sequence was found near the C-terminus of Cln3. Our data supports the hypothesis that Cln2 and Cln3 have distinct functions and locations, and the specificity of cyclin-dependent kinases is mediated in part by subcellular location.

scholarly journals Functional domains in nuclear import factor p97 for binding the nuclear localization sequence receptor and the nuclear pore.

1997 ◽  
Vol 8 (6) ◽  
pp. 945-956 ◽  
Author(s):  
N C Chi ◽  
S A Adam
Keyword(s):  
Nuclear Localization ◽  
Receptor Binding ◽  
Protein Import ◽  
Binding Domain ◽  
Nuclear Localization Sequence ◽  
Functional Domains ◽  
Nuclear Pore ◽  
Binding Domains ◽  
Localization Sequence ◽  
Ran Gtp

The interaction of the nuclear protein import factor p97 with the nuclear localization sequence (NLS) receptor, the nuclear pore complex, and Ran/TC4 is important for coordinating the events of protein import to the nucleus. We have mapped the binding domains on p97 for the NLS receptor and the nuclear pore. The NLS receptor-binding domain of p97 maps to the C-terminal 60% of the protein between residues 356 and 876. The pore complex-binding domain of p97 maps to residues 152-352. The pore complex-binding domain overlaps the Ran-GTP- and Ran-GDP-binding domains on p97, but only Ran-GTP competes for docking in permeabilized cells. The N-ethylmaleimide sensitivity of the p97 for docking was investigated and found to be due to inhibition of p97 binding to the pore complex and to the NLS receptor. Site-directed mutagenesis of conserved cysteine residues in the pore- and receptor-binding domains identified two cysteines, C223 and C228, that were required for p97 to bind the nuclear pore. Inhibition studies on docking and accumulation of a NLS protein provided additional evidence that the domains identified biochemically are the functional domains involved in protein import. Together, these results suggest that Ran-GTP dissociates the receptor complex and prevents p97 binding to the pore by inducing a conformational change in the structure of p97 rather than simple competition for binding sites.

scholarly journals Determination of the functional domains involved in nucleolar targeting of nucleolin.

1993 ◽  
Vol 4 (12) ◽  
pp. 1239-1250 ◽  
Author(s):  
L Créancier ◽  
H Prats ◽  
C Zanibellato ◽  
F Amalric ◽  
B Bugler
Keyword(s):  
Rna Binding ◽  
Chimeric Protein ◽  
Nuclear Localization Sequence ◽  
Binding Domains ◽  
Rich Domain ◽  
C Terminus ◽  
N Terminus ◽  
Localization Sequence ◽  
Nuclear Location

Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.

scholarly journals G Protein-Coupled Receptor Kinase 5 Contains a DNA-Binding Nuclear Localization Sequence

2004 ◽  
Vol 24 (23) ◽  
pp. 10169-10179 ◽  
Author(s):  
Laura R. Johnson ◽  
Mark G. H. Scott ◽  
Julie A. Pitcher
Keyword(s):  
Dna Binding ◽  
G Protein ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Calcium Ionophore ◽  
Nuclear Localization Sequence ◽  
G Protein Coupled Receptor ◽  
Gpcr Activation ◽  
Localization Sequence ◽  
G Protein Coupled

ABSTRACT G protein-coupled receptor kinases (GRKs) mediate desensitization of agonist-occupied G protein-coupled receptors (GPCRs). Here we report that GRK5 contains a DNA-binding nuclear localization sequence (NLS) and that its nuclear localization is regulated by GPCR activation, results that suggest potential nuclear functions for GRK5. As assessed by fluorescence confocal microscopy, transfected and endogenous GRK5 is present in the nuclei of HEp2 cells. Mutation of basic residues in the catalytic domain of GRK5 (between amino acids 388 and 395) results in the nuclear exclusion of the mutant enzyme (GRK5Δ NLS), demonstrating that GRK5 contains a functional NLS. The nuclear localization of GRK5 is subject to dynamic regulation. Calcium ionophore treatment or activation of Gq-coupled muscarinic-M3 receptors promotes the nuclear export of the kinase in a Ca2+/calmodulin (Ca2+/CaM)-dependent fashion. Ca2+/CaM binding to the N-terminal CaM binding site of GRK5 mediates this effect. Furthermore, GRK5, but not GRK5Δ NLS or GRK2, binds specifically and directly to DNA in vitro. Consistent with their presence in the nuclei of transfected cells, all the GRK4, but not GRK2, subfamily members contain putative NLSs. These results suggest that the GRK4 subfamily of GRKs may play a signaling role in the nucleus and that GRK4 and GRK2 subfamily members perform divergent cellular functions.

scholarly journals Phosphorylation and nuclear transit modulate the balance between normal function and terminal aggregation of the yeast RNA-binding protein Ssd1

2017 ◽  
Vol 28 (22) ◽  
pp. 3057-3069 ◽  
Author(s):  
Cornelia Kurischko ◽  
James R. Broach
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Export ◽  
Rna Binding ◽  
Binding Protein ◽  
Neurological Diseases ◽  
Rna Binding Protein ◽  
Normal Function ◽  
Nuclear Localization Sequence ◽  
P Bodies ◽  
Localization Sequence

Yeast Ssd1 is an RNA-binding protein that shuttles between the nucleus and cytoplasm. Ssd1 interacts with its target mRNAs initially during transcription by binding through its N-terminal prion-like domain (PLD) to the C-terminal domain of RNA polymerase II. Ssd1 subsequently targets mRNAs acquired in the nucleus either to daughter cells for translation or to stress granules (SGs) and P-bodies (PBs) for mRNA storage or decay. Here we show that PB components assist in the nuclear export of Ssd1and subsequent targeting of Ssd1 to PB sites in the cytoplasm. In the absence of import into the nucleus, Ssd1 fails to associate with PBs in the cytoplasm but rather is targeted to cytosolic insoluble protein deposits (IPODs). The association of Ssd1 either with IPOD sites or with PB/SG requires the PLD, whose activity is differentially regulated by the Ndr/LATS family kinase, Cbk1: phosphorylation suppresses PB/SG association but enhances IPOD formation. This regulation likely accrues from a phosphorylation-sensitive nuclear localization sequence located in the PLD. The results presented here may inform our understanding of aggregate formation by RBP in certain neurological diseases.

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