A novel conserved nuclear localization signal is recognized by a group of yeast importins

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin beta family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin alpha/beta complex are well known, the signals recognized by other importin beta-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x)(n)V/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.     

Response of Saccharomyces cerevisiae to ethanol stress involves actions of protein Asr1p

During the fermentation process of Saccharomyces cerevisiae, yeast cells must rapidly respond to a wide variety of external stresses in order to survive the constantly changing environment, including ethanol stress. The accumulation of ethanol can severely inhibit cell growth activity and productivity. Thus, the response to changing ethanol concentrations is one of the most important stress reactions in S. cerevisiae and worthy of thorough investigation. Therefore, this study examined the relationship between ethanol tolerance in S. cerevisiae and a unique protein called alcohol sensitive RING/PHD finger 1 protein (Asr1p). A real-time PCR showed that upon exposure to 8% ethanol, the expression of Asr1 was continuously enhanced, reaching a peak 2 h after stimulation. This result was confirmed by monitoring the fluorescence levels using a strain with a green fluorescent protein tagged to the C-terminal of Asr1p. The fluorescent microscopy also revealed a change in the subcellular localization before and after stimulation. Furthermore, the disruption of the Asr1 gene resulted in hypersensitivity on the medium containing ethanol, when compared with the wild-type strain. Thus, when taken together, the present results suggest that Asr1 is involved in the response to ethanol stress in the yeast S. cerevisiae.     

Central Role of the Oxygen-dependent Degradation Domain of Drosophila HIFα/Sima in Oxygen-dependent Nuclear Export

The Drosophila HIFα homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFα/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension.     

Hypertrophic cardiomyopathy-causing Asp175asn and Glu180gly Tpm1 mutations shift tropomyosin strands further towards the open position during the ATPase cycle

Yeast calmodulin known to be ubiquitylated in vivo in a Ca²⁺ dependent manner has long remained an orphan substrate. Here we identify Saccharomyces cerevisiae Asr1p as an ubiquitin E3 ligase for yeast calmodulin, a protein involved in calcium signaling. A short region within Asr1p-C harboring two putative calmodulin-binding motifs is sufficient and necessary for interaction with calmodulin. The interaction is direct, occurs in vivo and depends on physiological concentrations of Ca²⁺. A minimal set of purified proteins including Asr1p E3 ligase was sufficient for in vitro ubiquitylation of calmodulin, a reaction that required a functional Asr1p Ring domain. We propose a role of the Asr1p E3 ligase activity in coping with stress.     

Nuclear import of Upf3p is mediated by importin-alpha/-beta and export to the cytoplasm is required for a functional nonsense-mediated mRNA decay pathway in yeast

Upf3p, which is required for nonsense-mediated mRNA decay (NMD) in yeast, is primarily cytoplasmic but accumulates inside the nucleus when UPF3 is overexpressed or when upf3 mutations prevent nuclear export. Upf3p physically interacts with Srp1p (importin-alpha). Upf3p fails to be imported into the nucleus in a temperature-sensitive srp1-31 strain, indicating that nuclear import is mediated by the importin-alpha/beta heterodimer. Nuclear export of Upf3p is mediated by a leucine-rich nuclear export sequence (NES-A), but export is not dependent on the Crm1p exportin. Mutations identified in NES-A prevent nuclear export and confer an Nmd(-) phenotype. The addition of a functional NES element to an export-defective upf(-) allele restores export and partially restores an Nmd(+) phenotype. Our findings support a model in which the movement of Upf3p between the nucleus and the cytoplasm is required for a fully functional NMD pathway. We also found that overexpression of Upf2p suppresses the Nmd(-) phenotype in mutant strains carrying nes-A alleles but has no effect on the localization of Upf3p. To explain these results, we suggest that the mutations in NES-A that impair nuclear export cause additional defects in the function of Upf3p that are not rectified by restoration of export alone.     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.     

Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export

Pab1 is the major poly(A)-binding protein in yeast. It is a multifunctional protein that mediates many cellular functions associated with the 3'-poly(A)-tail of messenger RNAs. Here, we characterize Pab1 as an export cargo of the protein export factor Xpo1/Crm1. Pab1 is a major Xpo1/Crm1-interacting protein in yeast extracts and binds directly to Xpo1/Crm1 in a RanGTP-dependent manner. Pab1 shuttles rapidly between the nucleus and the cytoplasm and partially accumulates in the nucleus when the function of Xpo1/Crm1 is inhibited. However, Pab1 can also be exported by an alternative pathway, which is dependent on the MEX67-mRNA export pathway. Import of Pab1 is mediated by the import receptor Kap108/Sxm1 through a nuclear localization signal in its fourth RNA-binding domain. Interestingly, inhibition of Pab1's nuclear import causes a kinetic delay in the export of mRNA. Furthermore, the inviability of a pab1 deletion strain is suppressed by a mutation in the 5'-3' exoribonuclease RRP6, a component of the nuclear exosome. Therefore, nuclear Pab1 may be required for efficient mRNA export and may function in the quality control of mRNA in the nucleus.     

Cortical recruitment and nuclear-cytoplasmic shuttling of Scd5p, a protein phosphatase-1-targeting protein involved in actin organization and endocytosis

Scd5p regulates endocytosis and cortical actin organization as a targeting subunit for the Ser/Thr protein phosphatase-1 (PP1) in yeast. To identify localization signals in Scd5p required for cell surface recruitment, visualization of GFP-tagged Scd5 truncations and deletions was performed. Scd5p contains a PP1 binding site, a 3-repeat region of 20 amino acids (3R), and a 9-repeat region of 12 amino acids (9R). We found that the 9R is critical for cortical localization of Scd5p, but cortical recruitment is not essential for Scd5p's function in actin organization and endocytosis. We propose that Scd5p can target PP1 to endocytic factors in the cytoplasm that have been disassembled and/or inactivated by phosphorylation. We also found that Scd5p undergoes nuclear-cytoplasmic shuttling in a Crm1p-dependent manner. Scd5p-DeltaCT lacking the 9R region and its nuclear export signal (NES) accumulates in the nucleus, causing cortical actin and endocytic defects. Cytoplasmic localization and function of Scd5p-DeltaCT is restored by NES addition. However, removal of Scd5p's nuclear localization signal prevents nuclear entry, but endocytosis and actin organization remain relatively normal. These results indicate that nuclear-cytoplasmic shuttling is not required for regulation of Scd5p's cortical function and suggest that Scd5p has an independent nuclear function.     

Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha

Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and importin beta. Srp1p, the Saccharomyces cerevisiae homologue of importin alpha, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state. We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p. Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC). Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain. We show that Nup2p effectively releases the NLS protein from importin alpha-importin and beta and strongly binds to the importin heterodimer via Srp1p. Kap95p (importin beta) is released from this complex by a direct interaction with Gsp1p-GTP. These data suggest that besides Gsp1p, which disassembles the NLS-importin alpha-importin beta complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p. We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import. Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC. The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus.     

Asr1, an alcohol-responsive factor of Saccharomyces cerevisiae, is dispensable for alcoholic fermentation

Yeast Asr1 is the first reported protein whose intracellular distribution changes specifically in response to alcohol (Betz et al. (2004) J Biol Chem 279:28174–28181). It was reported that Asr1 is required for tolerance to alcohol and plays an important role in the alcohol stress response. Therefore, Asr1 is of interest to brewers and winegrowers attempting to improve the techniques of alcoholic fermentation. We verified the importance of Asr1 in the alcohol stress response during alcoholic fermentation. Although we reconfirmed the alcohol-responsive changes in the intracellular localization of Asr1, we could not detect the effects of Asr1-deficiency on Japanese sake brewing or winemaking. In addition, we could not reconfirm the hypersensitivity of Asr1-deficient mutants to alcohol and sodium dodecyl sulfate. Instead, we conclude that Asr1 is not required and nor important for tolerance to alcohol stress.     

Cytoplasmic localization of Wis1 MAPKK by nuclear export signal is important for nuclear targeting of Spc1/Sty1 MAPK in fission yeast

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1(+) does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.     

Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores.

An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.     

The carrier Msn5p/Kap142p promotes nuclear export of the hsp70 Ssa4p and relocates in response to stress

Cytoplasmic hsp70s like yeast Ssa4p shuttle between nucleus and cytoplasm under normal growth conditions but accumulate in nuclei upon stress. This nuclear accumulation is only transient, and Ssa4p relocates to the cytoplasm when cells recover. We show here that Ssa4p nuclear export is independent of Xpol/Crm1 and identify the importin-beta family member Msn5p/Kap142p as the exporter for Ssa4p. In growing cells and in vitro, Msn5p and Ssa4p generate genuine export complexes that require Ran/Gsp1p-GTP. Furthermore, nucleoporin Nup82p, which plays a role in Msn5p-mediated transport, is necessary for efficient export of Ssa4p. In living cells, stress not only regulates Ssa4p localization, but also controls the distribution of Msn5p. Msn5p is concentrated in nuclei of unstressed cells, but appears in the cytoplasm upon exposure to ethanol, heat, starvation or severe oxidative stress. In addition, growth on non-fermentable carbon sources relocates a portion of Msn5p to the cytoplasm and leads to a partial nuclear accumulation of Ssa4p. Taken together, growth and stress conditions that localize the transporter Msn5p to the cytoplasm also induce the nuclear accumulation of its cargo Ssa4p.     

Nuclear import and export signals enable rapid nucleocytoplasmic shuttling of the atypical protein kinase C lambda

The atypical protein kinase C (PKC) isoenzymes, lambda/iota- and zetaPKC, play important roles in cellular signaling pathways regulating proliferation, differentiation, and cell survival. By using green fluorescent protein (GFP) fusion proteins, we found that wild-type lambdaPKC localized predominantly to the cytoplasm, whereas both a kinase-defective mutant and an activation loop mutant accumulated in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal part of the zinc finger domain of lambdaPKC. Leptomycin B treatment induced rapid nuclear accumulation of GFP-lambda as well as endogenous lambdaPKC suggesting the existence of a CRM1-dependent nuclear export signal (NES). Consequently, we identified a functional leucine-rich NES in the linker region between the zinc finger and the catalytic domain of lambdaPKC. The presence of both the NLS and NES enables a continuous shuttling of lambdaPKC between the cytoplasm and nucleus. Our results suggest that the exposure of the NLS in both lambda- and zetaPKC is regulated by intramolecular interactions between the N-terminal part, including the pseudosubstrate sequence, and the catalytic domain. Thus, either deletion of the N-terminal region, including the pseudosubstrate sequence, or a point mutation in this sequence leads to nuclear accumulation of lambdaPKC. The ability of the two atypical PKC isoforms to enter the nucleus in HeLa cells upon leptomycin B treatment differs substantially. Although lambdaPKC is able to enter the nucleus very rapidly, zetaPKC is much less efficiently imported into the nucleus. This difference can be explained by the different relative strengths of the NLS and NES in lambdaPKC compared with zetaPKC.     

Nucleocytoplasmic Shuttling of the TACC Protein Mia1p/Alp7p Is Required for Remodeling of Microtubule Arrays during the Cell Cycle

Microtubule arrays are remodeled as cells proceed through the cell cycle. It is important to understand how remodeling is regulated in time and space. In fission yeast, the conserved microtubule associated TACC/TOG complex plays an important role in organizing microtubules throughout the cell cycle. Here we show that this complex undergoes nucleocytoplasmic shuttling through the nuclear import and export signals located in the TACC protein Mia1p/Alp7p. When the Crm1p-dependent nuclear export signal of Mia1p is disabled, Mia1p accumulates in the nucleus while its partner protein Alp14p/TOG is restricted to the cytoplasm. This leads to defects in assembly of both interphase arrays and the mitotic spindle. Artificial targeting of Alp14p to the nucleus partially rescues the mitotic spindle defects caused by lack of Mia1p nuclear export. Interestingly, the nuclear export sequence of Mia1p appears to overlap with the Alp14p binding site. We propose that intricate regulation of the subcellular distribution of TACC/TOG complexes drives microtubule array remodeling as cells progress through the cell cycle.     

hGTSE-1 expression stimulates cytoplasmic localization of p53

hGTSE-1 (human G(2) and S phase-expressed-1) is a cell cycle-regulated protein mainly localized in the cytoplasm and apparently associated with the microtubules. hGTSE-1 is able to down-regulate levels and activity of the p53 tumor suppressor protein: it binds the C-terminal region of p53 and represses its ability to induce apoptosis after DNA damage. Here we report that, after DNA damage, hGTSE-1 becomes stabilized in a p53-independent way and accumulated in the nucleus. Further characterization of hGTSE-1 localization revealed increased nuclear staining in unstressed cells after treatment with the nuclear export inhibitor leptomycin B, or when a nuclear export signal (NES) located in its C-terminal region was mutated. Finally, we provide evidence that hGTSE-1 ectopic expression, in addition to p53 protein levels down-regulation, is able to enhance cytoplasmic localization of p53. Interestingly, NES-mutated hGTSE-1 accumulates in the nucleus, binds p53 but looses its ability to enhance cytoplasmic redistribution of p53 and to regulate p53 protein levels. Similarly, when wild type hGTSE-1 functions on p53 were analyzed in cells lacking Mdm2, it failed in regulating both p53 localization and protein levels, thus indicating that hGTSE-1 requires an intact NES and functional Mdm2 for the regulation of p53. Our results provide new insights into the mechanism of hGTSE-1 function, whereby its characterized nucleo-cytoplasmic shuttling ability is required to regulate p53.