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At least 37 records · Page 2Linked to original sources
PubMed2004

Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals.

The interaction between phosphatidylserine and its receptor on phagocytic cells plays a critical role in the clearance of apoptotic bodies under normal physiological condition. A specific receptor for phosphatidylserine (PSR) has recently been identified by phage display and shown to mediate phosphatidylserine dependent phagocytosis. Here we show that the protein encoded by the PSR cDNA is localized in the nuclei through multiple nuclear localization signals. First, a fusion between PSR and GFP is localized in the nuclei of transfected cells, suggesting that PSR have intrinsic nuclear localization capability. Indeed, affinity-purified anti-PSR antibodies identified a 47 kDa protein species in cells transfected with untagged PSR and localized this protein in the nuclei by immunofluorescent confocal microscopy. In NIH3T3 cells, which express endogenous PSR mRNA, a similar 47 kDa species was detected and localized in the nuclei. Finally, multiple nuclear localization signals were identified in PSR sequence, each capable of targeting GFP to the nuclei. Together, these results suggest that PSR may serve a dual role both on the cell surface and in the nuclei.

Adenocarcinoma↗
PubMed2001

Senescence represses the nuclear localization of the serum response factor and differentiation regulates its nuclear localization with lineage specificity.

The differentiation of cultured 3T3T mesenchymal stem cells into adipocytes represses growth factor responsiveness by limiting the nuclear localization of the serum response factor (SRF) that binds to and activates the promoters of growth control genes that contain the serum response elements (SRE), such as junB and c-fos. The regulation of SRF nuclear localization by adipocyte differentiation is specific, because we show that adipocyte differentiation does not repress the nuclear localization of six other transacting factors. To determine if repression of growth factor responsiveness that occurs during senescence also represses the nuclear localization of SRF, we studied normal human WI-38 fibroblasts at low versus high population doublings. The results show that SRF localizes to the nucleus of proliferative cells whereas in senescent cells SRF can not be detected in the nucleus. This result is apparent in both immunofluorescence assays and in western blot analysis. We next evaluated the cellular distribution of SRF in selected human tissues to determine whether the loss of proliferative potential in vivo could have a different effect on SRF nuclear localization. We found that in cells of the small bowel mucosa, differentiation modulates SRF nuclear localization in an opposite manner. Minimal SRF expression and nuclear localization is evident in undifferentiated cells at the base of crypts whereas increased SRF expression and nuclear localization is evident in differentiated cells at the surface tip of the villus. These results together establish that regulation of SRF expression and nuclear localization is important in senescence and differentiation in a lineage specific manner.

Adipocytes↗
PubMed2006

Nuclear localization of PTEN by a Ran-dependent mechanism enhances apoptosis: Involvement of an N-terminal nuclear localization domain and multiple nuclear exclusion motifs.

The targeting of the tumor suppressor PTEN protein to distinct subcellular compartments is a major regulatory mechanism of PTEN function, by controlling its access to substrates and effector proteins. Here, we investigated the molecular basis and functional consequences of PTEN nuclear/cytoplasmic distribution. PTEN accumulated in the nucleus of cells treated with apoptotic stimuli. Nuclear accumulation of PTEN was enhanced by mutations targeting motifs in distinct PTEN domains, and it was dependent on an N-terminal nuclear localization domain. Coexpression of a dominant negative Ran GTPase protein blocked PTEN accumulation in the nucleus, which was also affected by coexpression of importin alpha proteins. The lipid- and protein-phosphatase activity of PTEN differentially modulated PTEN nuclear accumulation. Furthermore, catalytically active nuclear PTEN enhanced cell apoptotic responses. Our findings indicate that multiple nuclear exclusion motifs and a nuclear localization domain control PTEN nuclear localization by a Ran-dependent mechanism and suggest a proapoptotic role for PTEN in the cell nucleus.

3T3 Cells↗
PubMed2004

The SV40 T antigen nuclear localization sequence enhances nuclear import of vector DNA in embryos of a crustacean (Litopenaeus schmitti).

A genetic transformation system for penaeid shrimp could provide a powerful technique for the improvement of different production traits of importance for a sustainable aquaculture. The development of a successful transformation system depends on the ability to efficiently introduce exogenous DNA into the target species. The ability of the nuclear localization signal (NLS) peptide of the SV40 T antigen to facilitate nuclear import and transient gene expression is known from vertebrate systems and for the first time, is shown here to be efficient in a crustacean species, i.e. the shrimp Litopenaeus schmitti. Electroporation was used to introduce the pCMV-lacZ plasmid that contains the human cytomegalovirus promoter/enhancer (CMV) fused to the beta-galactosidase (lacZ) coding region, into L. schmitti zygotes. Supercoiled DNA was used at 50 or 500 ng/microl naked or bound to NLS peptide. The hatching rate of electroporated zygotes was around 60% for all groups, except from the pCMV-lacZ:NLS group at 500 ng/microl (43%). Based on Southern blot analyses of polymerase chain reaction (PCR) products the gene transfer frequency was 2-fold higher using DNA:NLS complexes than with naked DNA (23.8% vs. 11.5%, with 50 ng/microl of plasmid DNA, 44.3% vs. 28.8% with 500 ng/microl). The beta-galactosidase activity assay indicated that nuclear uptake is faster for the DNA:NLS complexes than for naked DNA. The beta-galactosidase activity was always higher in the DNA:NLS groups than in the naked DNA groups. To our knowledge, this is the first report on the use of an NLS peptide to improve gene transfer and nuclear uptake in crustaceans.

Active Transport, Cell Nucleus↗
PubMed2005

Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles.

The nuclear membrane is a tight barrier against the delivery of therapeutic genes into non-dividing tissue cells. Overcoming this barrier with the aid of peptidic nuclear localization signals (NLS) is crucial for improving the performance of synthetic gene-delivery vehicles. In this article, we examine the nuclear transport of lambda phage particles displaying various peptides containing the minimum NLS of SV40 T antigen on their surface. As the minimum NLS (PKKKRKV) is a binding domain to importin alpha, recombinant proteins and molecular conjugates containing this peptide accumulate into the nucleus efficiently. However, we find that the C-terminal and N-terminal structures besides the minimum NLS profoundly affect the efficiency of the nuclear transport of the phage particles as well as their binding capacity to importin alpha: either truncation of a few amino acid residues from the C-terminus or the replacement of the N-terminus with a FLAG- or c-myc-tag abolish both of these biological activities. The structure of the optimized NLS is unpredictable from conventional protein transport assay and from the structural analysis in silico. Our results reveal that the objects with 50 nm in diameter can pass through the nuclear pore complex when the optimized NLS is displayed at a sufficient density on their surface.

Active Transport, Cell Nucleus↗
PubMed1997

SV40 large tumor antigen nuclear import is regulated by the double-stranded DNA-dependent protein kinase site (serine 120) flanking the nuclear localization sequence.

Nuclear localization sequence (NLS)-dependent nuclear import of SV40 large tumor antigen (T-Ag) fusion proteins is regulated by phosphorylation sites for casein kinase II (CKII) and the cyclin-dependent kinase Cdc2 amino-terminal to the NLS (amino acids 126-132). Between the T-Ag CKII and Cdc2 sites is a site (Ser120) for the double-stranded DNA-dependent protein kinase (dsDNA-PK), which we show here for the first time to play a role in regulating T-Ag nuclear import. We replaced Ser120 by aspartic acid or alanine using site-directed mutagenesis and assessed the effects on nuclear transport kinetics both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in HTC rat hepatoma cells. Maximal nuclear accumulation of the Asp120 and Ala120 protein derivatives was approximately 40% and 70% reduced in vivo, respectively, compared with that of the wild type protein, and similarly reduced in vitro, although to a lesser extent. This implies that the dsDNA-PK site regulates the maximal level of nuclear accumulation, normally functioning to enhance T-Ag nuclear transport; the higher accumulation of the Asp120 protein compared with the Ala120 protein indicates that negative charge at the dsDNA-PK site is mechanistically important in regulating nuclear import. The Asp120 protein accumulated in the nucleus at a faster rate than the wild type protein, implying that phosphorylation at Ser120 may also regulate the nuclear import rate. CKII phosphorylation of the Asp120 protein in cytosol or by purified CKII was approximately 30% higher than that of the Ser120 and Ala120 proteins, while negative charge at the CKII site increased dsDNA-PK phosphorylation of Ser120 by approximately 80% compared with wild type, implying physical and functional interactions between the two phosphorylation sites. Quantitation of NLS recognition by the importin 58/97 subunits using an enzyme-linked immunosorbent assay indicated that while the Ala120 protein derivative had a binding affinity very similar to that of wild type, the Asp120 derivative showed 40% higher affinity. In vitro CKII phosphorylation increased importin binding by about 30% in all cases. These results imply that negative charge at the dsDNA-PK site may enhance nuclear import through increasing both NLS recognition by importin subunits, and phosphorylation at the CKII site, which itself also facilitates NLS recognition by importin 58/97.

Amino Acid Sequence↗
PubMed1999

Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV.

The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333-374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.

Amino Acid Sequence↗
PubMed1999

Enhancement of polylysine-mediated transferrinfection by nuclear localization sequences: polylysine does not function as a nuclear localization sequence.

Polylysine (pLy) has been used successfully as a DNA carrier in receptor-mediated gene transfer, enhancement of transfection having been proposed to be in part through efficient nuclear targeting stemming from the resemblance of pLy to the nuclear localization sequence (NLS) from simian virus SV40 large tumor antigen (T-ag). In this study we test whether pLy carrying covalently attached peptides comprising the T-ag NLS (the pLyP101 derivative) can enhance transferrin-pLy-mediated transfection ("transferrinfection"). Unlike pLy itself or a pLy derivative (pLyP101T) carrying cross-linked T-ag NLS mutant peptides, pLyP101 significantly enhanced transferrinfection of a beta-galactosidase-expressing reporter plasmid. The basis of this was shown to be the ability of the pLyP101-plasmid DNA complex to be recognized with high affinity by the NLS-binding importin subunits, in contrast to pLyP101T- and pLy-plasmid complexes. Confocal laser scanning microscopy was used to determine the nuclear import kinetics of fluorescently labeled pLyP101 and pLyP101T in the presence of complexed plasmid, indicating that pLyP101 and not pLyP101T complexes accumulated rapidly in the nucleus. We conclude that pLy itself does not function as an NLS and that the addition of exogenous NLSs conferring interaction with the cellular nuclear import machinery can increase transferrinfection by enhancing the nuclear targeting of pLy-DNA complexes.

Amino Acid Sequence↗
PubMed1998

Human immunodeficiency virus type 1 Vpr localization: nuclear transport of a viral protein modulated by a putative amphipathic helical structure and its relevance to biological activity.

Protein import into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly clear that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Human immunodeficiency virus type 1 (HIV-1) Vpr is one such protein which does not have an identifiable canonical NLS and yet efficiently localizes to the nuclear compartment. Here, we use confocal microscopy to demonstrate that mutations in the putative central hydrophobic helix of Vpr result in the retention of the protein in highly localized ring-like structures around the nuclear periphery with striking impairment in their ability to enter the nuclear interior. By characterizing other biological activities associated with this protein, such as its ability to incorporate into budding virions and its ability to arrest cells in G2, we show that this helical domain is specific for the nuclear translocation of the protein with very little effect on these other functions. Interestingly, however, perturbation of this helical motif also perturbs the protein's ability to augment viral replication in primary human macrophages indicating that the integrity of this secondary structure is essential for optimal infection in these non-dividing cells.

Amino Acid Sequence↗
PubMed2005

Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals.

Phytochromes are red and far-red photoreceptors that regulate plant growth and development in response to environmental light cues. Phytochromes exist in two photo-interconvertible conformational states: an inactive Pr form and an active Pfr form. The alteration of phytochromes' subcellular location functions as a major regulatory mechanism of their biological activities. Whereas phytochromes in the Pr form localize in the cytoplasm, phytochromes in the Pfr form accumulate in the nucleus, where they interact with transcription factors to regulate gene expression. The molecular details of the regulation of phytochrome translocation by light are poorly understood. Using Arabidopsis phyB as a model, we demonstrate that the C-terminal PAS-related domain (PRD) is both necessary and sufficient for phyB nuclear import and that the entire C terminus is required for nuclear-body (NB) localization. We also show that phyB's N-terminal bilin lyase domain (BLD) and PHY domain interact directly with the PRD in a light-dependent manner. In vivo localization studies indicate that BLD-PHY is sufficient to regulate phyB's nuclear accumulation. For phyB nuclear localization, our results suggest a molecular mechanism in which the nuclear-localization signal in the PRD is masked by interactions with phyB's chromophore-attachment domains and unmasked by light-dependent conformational changes.

Arabidopsis↗
PubMed1992

The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells.

Here we show that the VirD2 protein of A. tumefaciens functions as a nuclear localizing protein in plant cells. The nuclear localization signal of VirD2 consists of two regions containing 4-5 basic amino acids (KRPR and RKRER), located within the C-terminal 34 amino acids. These regions conform to the KR/KXR/K motif required for numerous nuclear localized nonplant eukaryotic proteins. Each region independently directs a beta-glucuronidase reporter protein to the nucleus; however, both regions are necessary for maximum efficiency. VirD2 has been shown to be tightly bound to the 5' end of the single-stranded DNA transfer intermediate, T-strand, transferred from Agrobacterium to the plant cell genome. The present results imply that T-strand transport to the plant nucleus is mediated by the tightly attached VirD2 protein via an import pathway common to higher eukaryotes.

Agrobacterium tumefaciens↗
PubMed1995

Twin autonomous bipartite nuclear localization signals direct nuclear import of GT-2.

GT-2 is a DNA-binding protein with high target-sequence specificity toward functionally defined, positively acting cis elements in the rice phytochrome A gene promoter. Using immunocytochemical procedures, it is shown here that GT-2 is localized to the nucleus, consistent with a function in transcriptional regulation. Immunoblot and immunocytochemical analyses show that rice shoots contain higher levels of GT-2 protein than roots, and that no photo-induced changes in GT-2 abundance or spatial distribution are detectable in these tissues, a result consistent with the proposed constitutive activity of GT-2. In both shoots and roots, GT-2 protein is undetectable in meristematic tissue but becomes expressed at later stages of cellular development, consistent with a role in contributing to the pattern of phytochrome A gene expression. By transfecting protoplasts with a series of constructs containing deletion derivatives of GT-2 fused to beta-glucuronidase (GUS), followed by in situ localization of GUS activity, two independent, functionally active nuclear localization sequences (NLSs) have been identified in GT-2. One NLS resides within each of a pair of previously identified, spatially separate, trihelix motifs in the protein. Sequence inversion and alanine-scanning mutagenesis has identified residues within these NLSs necessary for nuclear localization. Each NLS contains two basic domains separated by 10 amino acids, conforming to the bipartite class of NLS involved in the targeting of numerous other nuclear localized proteins.

Amino Acid Sequence↗
PubMed2005

An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium.

Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD (171EDVSRFIKGKLLQKQQKIYKDLERF195) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues 48KKSYQDPEIIAHSRPRK64 that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to 48EF49 abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the 48EF49 construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.

Active Transport, Cell Nucleus↗
PubMed2002

An N-terminal truncated form of Orp150 is a cytoplasmic ligand for the anti-proliferative mushroom Agaricus bisporus lectin and is required for nuclear localization sequence-dependent nuclear protein import.

Nuclear localization sequence-dependent nuclear protein import is essential for maintaining cell function and can be selectively blocked in epithelial cells by mushroom (Agaricus bisporus) lectin. Here we report that a major intracellular ligand for this lectin is an N-terminally truncated form of oxygen-regulated protein 150 (Orp150), which lacks the endoplasmic reticulum translocation signal peptide of full-length Orp150. This cytoplasmic form of Orp150 expresses the lectin carbohydrate ligand (sialyl-2,3-galactosyl-beta1,3-N-acetylgalactosamine-alpha) and is shown to be essential for nuclear localization sequence-dependent nuclear protein import.

Active Transport, Cell Nucleus↗
PubMed1994

Genetically defined nuclear localization signal sequence of bovine papillomavirus E1 protein is necessary and sufficient for the nuclear localization of E1-beta-galactosidase fusion proteins.

The 605 amino acid E1 protein of bovine papillomavirus type 1 (BPV-1) is a multifunctional nuclear protein required for viral DNA replication. A nuclear localization signal (NLS) sequence was previously defined by point mutations in three short adjacent clusters of basic amino acids located in the amino-terminal region of the E1 protein. In this study, we used a fusion protein approach to evaluate the contribution of other regions of the E1 protein to nuclear transport. The nearly full-length E1 gene and six non-overlapping subfragments were each fused in-frame with the lacZ gene in a eukaryotic expression vector. Each clone was electroporated into COS-1 cells, and the intracellular location of the E1-beta-galactosidase fusion proteins was determined by immunofluorescence. Only the constructs containing the full-length E1 or a single subregion (E1-259; amino acids 84 to 166) produced fusion proteins that entered the nucleus. Point mutations in the NLS sequences of the E1-259-lacZ construct prevented nuclear translocation of the corresponding fusion protein. This confirms the previous result that the cluster of basic amino acids is critical for nuclear transport. Furthermore, the data obtained in this investigation indicated that the region of E1 containing the NLS sequence was not only necessary, but was also sufficient for nuclear localization. No other region of E1 contained independent nuclear localization activity.

Amino Acid Sequence↗
PubMed1999

Identification of a bipartite nuclear localization sequence necessary for nuclear import of 5-lipoxygenase.

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. This enzyme can reside either in the cytoplasm or the nucleus; its subcellular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fluorescence, similar to the distribution of native 5-lipoxygenase in primary alveolar macrophages. Within the 5-lipoxygenase protein is a sequence (Arg(638)-Lys(655)) that closely resembles a bipartite nuclear localization signal. Studies using deletion mutants indicated that this region was necessary for nuclear import of 5-lipoxygenase. Analysis of mutants containing specific amino acid substitutions within this sequence confirmed that it was this sequence that was necessary for nuclear import of 5-lipoxygenase and that a specific arginine residue was critical for this function. As nuclear import of 5-lipoxygenase may regulate leukotriene production, natural or induced mutations in this bipartite nuclear localization sequence may also be important in affecting leukotriene synthesis.

3T3 Cells↗
PubMed1995

Arginine-rich regions succeeding the nuclear localization region of the herpes simplex virus type 1 regulatory protein ICP27 are required for efficient nuclear localization and late gene expression.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein that localizes to the nuclei of infected cells. The strong nuclear localization signal (NLS) of ICP27 was identified recently and shown to reside in the amino-terminal portion of the polypeptide from residues 110 to 137 (W.E. Mears, V. Lam, and S.A. Rice, J. Virol. 69:935-947, 1995). There are also two arginine-rich regions directly succeeding the NLS. The first of these arginine-rich sequences (residues 141 to 151), together with the NLS, has been shown by Mears et al. to form the nucleolar localization signal. Arginine-rich motifs are common in domains involved in nuclear localization and RNA binding. To analyze the role of the arginine-rich regions in ICP27, we constructed stably transformed cell lines containing ICP27 mutants with deletions of all or parts of the NLS and arginine-rich regions. We also constructed mutants in which these regions were replaced with heterologous NLSs or RNA-binding domains. Characterization of these mutants indicated that the arginine-rich regions were required but not sufficient for wild-type localization of ICP27. More importantly, the NLS and arginine-rich regions were also essential to the function of ICP27. Mutants lacking these sequences were defective in late gene expression during infection even when ICP27 was properly localized to the nucleus by substitution of the NLS from simian virus 40 large T antigen. Further, the defect in late gene expression could not be overcome by replacement with the highly basic RNA-binding domain of human immunodeficiency virus type 1 Tat. The deficiency in late gene expression was independent of ICP27's role in stimulating viral DNA replication. In addition, localization of the HSV-1 proteins ICP4, ICP0, and ICP8 was unaffected by ICP27 mutants in this region. These results suggest that the arginine-rich regions are required for efficient nuclear localization and for the regulatory activity of ICP27 involved in viral late gene expression.

Amino Acid Sequence↗
PubMed1999

A nuclear export signal prevents Saccharomyces cerevisiae Hsp70 Ssb1p from stimulating nuclear localization signal-directed nuclear transport.

Hsp70 has been implicated in nuclear localization signal (NLS)-directed nuclear transport. Saccharomyces cerevisiae contains distinct SSA and SSB gene families of cytosolic Hsp70s. The nucleocytoplasmic localization of Ssa1p and Ssb1p was investigated using green fluorescent protein (GFP) fusions. Whereas GFP-Ssa1p localized both to the nucleus and cytoplasm, GFP-Ssb1p appeared only in the cytosol. The C-terminal domain of Ssb1p contains a leucine-rich nuclear export signal (NES) that is necessary and sufficient to direct nuclear export. The accumulation of GFP-Ssb1p in the nuclei of xpo1-1 cells suggests that Ssb1p shuttles across the nuclear envelope. Elevated levels of SSA1 but not SSB1 suppressed the NLS-GFP nuclear localization defects of nup188-Delta cells. Studies with Ssa1p/Ssb1p chimeras revealed that the Ssb1p NES is sufficient and necessary to inhibit the function of Ssa- or Ssb-type Hsp70s in nuclear transport. Thus, NES-less Ssb1p stimulates nuclear transport in nup188-Delta cells and NES-containing Ssa1p does not. We conclude that the differential function of Ssa1p and Ssb1p in nuclear transport is due to the NES-directed export of the Ssb1p and not to functional differences in their ATPase or peptide binding domains.

Adenosine Triphosphatases↗