Nucleocytoplasmic transport and nuclear envelope integrity in the fission yeast Schizosaccharomyces pombe

Methods 33 (2004) 226–238 www.elsevier.com/locate/ymeth

Nucleocytoplasmic transport and nuclear envelope integrity in the fission yeast Schizosaccharomyces pombe Minoru Yoshidaa,b and Shelley Sazerc,* a

c

Chemical Genetics Laboratory, RIKEN, Saitama 351-0198, Japan b CREST Research Project, JST, Saitama 332-0012, Japan Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA Accepted 17 November 2003 Available online 20 February 2004

Abstract The nuclear envelope is essential for compartmentalizing the nucleus from the cytoplasm in all eukaryotic cells. There is a tremendous flux of both RNA and proteins across the nuclear envelope, which is intact throughout the entire cell cycle of yeasts but breaks down during mitosis of animal cells. Transport across the nuclear envelope requires the recognition of cargo molecules by receptors, docking at the nuclear pore, transit through the nuclear pore, and then dissociation of the cargo from the receptor. This process depends on the RanGTPase system, transport receptors, and the nuclear pore complex. We provide an overview of the nuclear transport process, with particular emphasis on the fission yeast Schizosaccharomyces pombe, including strategies for predicting and experimentally verifying the signals that determine the sub-cellular localization of a protein of interest. We also describe a variety of reagents and experimental strategies, including the use of mutants and chemical inhibitors, to study nuclear protein import, nuclear protein export, nucleocytoplasmic protein shuttling, and mRNA export in fission yeast. The RanGTPase and its regulators also play an essential transport independent role in nuclear envelope re-assembly after mitosis in animal cells and in the maintenance of nuclear envelope integrity at mitosis in S. pombe. Several experimental strategies and reagents for studying nuclear size, nuclear shape, the localization of nuclear pores, and the integrity of the nuclear envelope in living fission yeast cells are described. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Schizosaccharomyces pombe; Fission yeast; Nucleocytoplasmic transport; RanGTPase; Nucleus; Nuclear envelope; Nuclear pore; Leptomycin B; pim1; crm1

1. Introduction 1.1. The nuclear envelope The nucleus and the cytoplasm are separated from one another by the nuclear envelope (NE), which is an impermeable barrier to the exchange of macromolecules. This means that most nuclear encoded RNA and certain protein complexes, such as the ribosome, must be transported from the nucleus to the cytoplasm and all nuclear proteins must be transported from their site of synthesis in the cytoplasm into the nucleus. The nuclear pore complex (NPC), a 125 million Dalton multi-protein * Corresponding author. Fax: 1-713-796-9438. E-mail address: [email protected] (S. Sazer).

1046-2023/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2003.11.018

structure, spans the nuclear envelope. It forms a channel through which ions and small molecules, including proteins <50 kDa, can diffuse and larger molecules must be selectively transported (reviewed in [1]). 1.2. The RanGTPase system The RanGTPase system is essential for the proper intracellular localization of RNA and protein in eukaryotic cells [2]. Ran is a small GTPase that is remarkably well conserved amongst eukaryotic organisms. The direct regulators of Ran (Spi1p in Schizosaccharomyces pombe), the Ran guanine nucleotide exchange factor (RanGEF) (Pim1p in S. pombe), the RanGTPase activating protein (RanGAP) (Rna1p in S. pombe), Ran binding protein 1 (RanBP1) (Sbp1p in

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S. pombe), and the guanine nucleotide dissociation factor Mog1p (Mog1p in S. pombe) are less well conserved in amino acid sequence but have the same biochemical properties (reviewed in [3]). These proteins interact with one another, and with other components of the transport system both biochemically and genetically, so it is likely that the Ran system participates in nucleocytoplasmic transport in S. pombe in the same way that it does in animal cells and budding yeast. For reasons that are not clear, loss of transport function when most of these proteins are mutated has not been demonstrated experimentally (see Section 1.6.2 for details). Altering the functioning of the RanGTPase and its regulators, by mutation or overexpression, causes an identical terminal phenotype in S. pombe. The cells progress normally through the cell cycle, initiate mitosis, but then arrest with hypercondensed separated chromosomes, a wide medial septum, cytoplasmic microtubules, and fragmented nuclear envelopes [4–7]. Therefore, whether Ran accumulates in the GDP- or GTP-bound form, the consequences to the cell are the same, suggesting that Ran must cycle between the GDPand GTP-bound forms for proper function. 1.3. RanGTP gradients The intracellular localization of Ran and its regulators is essential to their regulatory function. RanGEF is nuclear and chromatin associated, RanGAP is cytoplasmic, and RanGTPase is predominantly but not exclusively nuclear (reviewed in [8]). This asymmetric localization should result in a relatively high concentration of RanGTP in the nucleus and a relatively high concentration of RanGDP in the cytoplasm. This RanGTP gradient across the nuclear envelope has been observed in nuclei assembled in vitro [9] and is essential for nucleocytoplasmic transport in animal cells [1,10]. Transport of macromolecules across the nuclear envelope requires recognition of either the nuclear localization signal (NLS) or nuclear export signal (NES) on the cargo protein by a transport receptor, translocation of this complex to the nuclear pore, passage through the pore, and then release of the cargo in the appropriate cellular compartment (reviewed in [1,10]). Ran regulates the stability of the cargo–receptor complexes in both the nucleus and the cytoplasm (see Fig. 1). A protein destined for import into the nucleus forms a stable complex with its import receptor in the cytoplasm, where the concentration of RanGTP is low. Once in the nucleus RanGTP binds to the import receptor and causes a conformational change that destabilizes the complex and dissociates the import cargo from its receptor in the nucleus. Conversely, a protein or protein/RNA complex destined for export forms a stable complex with both an export receptor and RanGTP inside of the nucleus, but the complex dissociates in the cytoplasm where

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RanGTP is hydrolyzed by RanGAP. Although several models have been proposed, the mechanism by which cargo passes through the pore itself is still unclear. Because RanGEF is chromatin-associated, RanGTP may be enriched in the vicinity of the condensed mitotic chromosomes. This chromosome-based gradient of RanGTP should be steepest in mitotic animal cells after nuclear envelope breakdown and has been demonstrated in vitro in a Xenopus laevis cell free system [9]. It has been proposed that this RanGTP gradient is essential for both spindle formation at mitotic onset and nuclear envelope reformation after mitosis in animal cells [11]. While it is not clear whether such a gradient exists in yeast, the RanGTPase is required for both nuclear envelope integrity [6] and spindle formation [12,13] in S. pombe. 1.4. Transport receptors 1.4.1. Importins 1.4.1.1. Importin-b. The importin-b family of transport receptors carry cargoes both into and out of the nucleus and are called importins (karyopherins) or exportins, respectively. Most of them are capable of binding directly to cargo and transporting it across the NE, while others require an importin-a adaptor (see below) [2]. The importin-b subunit of the receptor/cargo complex facilitates docking at the nuclear pore by directly binding to nucleoporins [2]. Import complexes are destabilized by the binding of RanGTP to the importin-b subunit whereas export complexes are stabilized by RanGTP binding. Based on sequence analysis there are 13 predicted importin-b proteins in the S. pombe genome [14]. 1.4.1.2. Importin-a. Importin-a is a transport adaptor that recognizes and directly binds to the classical NLS of proteins destined for nuclear import. The importin-a/ cargo complex then associates with an importin-b protein, which mediates the import process as described above. In S. pombe, there is one importin-a homologue, Cut15p [15], and an as yet uncharacterized importin-a homologue named imp1 [14]. 1.4.2. Exportins 1.4.2.1. Crm1. Crm1p is a member of the importin-b family that is responsible for the nuclear export of proteins containing a leucine-rich NES (NES) [16]. The export activity of Crm1 can be inhibited by leptomycin B (LMB) [17] or by a temperature-sensitive or coldsensitive mutation in crm1 [18,19]. LMB produced by Streptomyces sp. ATS1287 was originally discovered in a screen for anti-fungal antibiotics. LMB is an unsaturated long-chain fatty acid with a terminal d-lactone ring. S. pombe, unlike Saccharomyces

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Fig. 1. General scheme of nucleocytoplasmic transport. An import cargo and receptor form a stable complex in the cytoplasm where the concentration of RanGTP is low. This complex is destabilized in the nucleus by the binding of RanGTP. An export cargo and receptor form a stable trimeric complex in the nucleus with RanGTP and this complex is destabilized in the cytoplasm upon the hydrolysis of RanGTP to RanGDP.

Fig. 2. The intracellular localization of Pap1p can be used to monitor both nuclear protein import and export. Pap1p shuttles between the nucleus and the cytoplasm in wild-type cells. When treated with hydrogen peroxide, nuclear export is blocked and Pap1 accumulates in the nucleus. After removal of hydrogen peroxide, the protein re-localizes to the cytoplasm. Pap1p can be used as a reporter to monitor defects in protein import and in protein export.

Fig. 3. The nuclear export of GFP-Pap1p can be blocked by treatment with LMB or hydrogen peroxide. (A) Wild-type cells expressing GFP-Pap1p, which is primarily cytoplasmic at steady state. (B) Wild-type cells expressing GFP-Pap1p treated with LMB accumulate the protein in the nucleus. (C) Wild-type cells expressing GFP-Pap1p treated with hydrogen peroxide accumulate the protein in the nucleus.

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cerevisiae, is highly sensitive to LMB [20]. The molecular target of LMB was identified using fission yeast genetics to isolate a LMB-resistant mutant of S. pombe that was found to be mutated in crm1þ . crm1 was originally identified as a gene essential for chromosome region maintenance [18]. It encodes a 115 kDa protein that is essential for proliferation of S. pombe and is localized in the nucleus and its periphery [18]. Since the cellular and biochemical phenotypes of the cold-sensitive mutant crm1-809 were identical to those of wild-type cells treated with LMB, Crm1p or its regulatory cascade was proposed to be the cellular target of LMB [21]. An important clue to unraveling the Crm1 function was provided by Wolff et al. [22], who reported that LMB inhibited nuclear export of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Revdependent mRNA. Eventually several groups showed that the mammalian homologue of Crm1 (CRM1/ exportin 1) acts as the leucine-rich NES export receptor [16,23,24]. LMB was shown to inhibit the Crm1-mediated nuclear export of NES-bearing proteins by binding covalently to the single cysteine residue at Cys529 in the central conserved region of S. pombe Crm1 [25]. The single amino acid change from Cys-529 to Ser in Crm1 is sufficient to confer high LMB resistance to wild-type S. pombe. Interestingly, Cys-529 is conserved in LMB-sensitive organisms such as humans, but not in LMB-insensitive organisms such as S. cerevisiae (Thr-539). On the other hand, nuclear import of NLSbearing proteins is not affected by LMB treatment. By using biotinylated LMB as a probe, the only protein bound to LMB was Crm1. Thus, LMB serves as a tool with extremely high specificity for studies of nucleocytoplasmic transport. Several nucleocytoplasmic shuttling proteins in S. pombe, including Dsk1 [16] , Pap1 [26], Cdc25 [27], Mei2 [28], and Ste11 [29], have been shown, using LMB, to be cargoes for Crm1. Of these proteins, the Pap1 NES is unique in that it contains cysteine residues that are responsible for sensing oxidative stress [25,30,31]. 1.4.2.2. Cse1. Cse1p is a member of the importin-b family that exports importin-a to the cytoplasm [2]. This receptor recycling function is essential for continued nucleocytoplasmic transport. There is one Cse1-like protein in the S. pombe genome [14]. 1.5. Transport cargo 1.5.1. Predicting whether a protein has an NLS There are two well-characterized types of nuclear localization signals in higher eukaryotic proteins, that are referred to as ‘‘classical’’ NLSs. The SV40 large T antigen type NLS (PKKKRKV) consists of a short stretch of basic amino acids preceded by a helix breaking residue. The bipartite type NLS, first characterized

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in the nucleoplasmin protein (KRPAAIKKAGQA KKKK), consists of two clusters of basic amino acids separated by any 9–12 amino acids (reviewed in [1]). Both of these classical NLSs function in S. pombe to direct proteins into the nucleus [12,32,33]. Although these two types of classical NLSs do not have a common consensus sequence, they are both recognized by importin-a [34]. There are also nuclear proteins that have neither of these types of NLS sequences and may carry a less well-defined ‘‘non-classical’’ NLS that is directly recognized by an importin-b receptor. Using a proteinÕs amino acid sequence to predict whether it is likely to be nuclear is difficult for several reasons: (1) commonly used database searches can identify patches of basic amino acids, and thereby classical NLSs, but cannot identify non-classical NLSs; (2) finding a signature motif for one of these classical NLSs in a protein is not a guarantee that the protein is nuclear, since many non-nuclear proteins contain patches of basic amino acids; (3) proteins may have several consensus NLS sequences, not all of which are functional; and (4) proteins lacking an NLS may enter the nucleus by binding to an NLS containing protein. Commonly used methods to identify a potential NLS in a protein include searching for homologues with known NLSs in sequence databases like SWISS-PROT and looking for known signal sequences in motif databases like PROSITE. However, both these methods fail to identify a vast majority of NLS signals [35]. Based on these analyses a computational method was developed that identifies putative NLSs in proteins with high accuracy. Dr. Rajesh Nair, of the Columbia University Bioinformatics Center (CUBIC), has recently used this approach to analyze the identified ORFs in the S. pombe genome and compiled a list of nearly 400 putative nuclear proteins, which can be accessed at his web site: http://cubic.bioc.columbia.edu/cgi/var/nair/predictNLS/ Genome.pl. 1.5.2. Predicting whether a protein has an NES Nuclear export signals are typically leucine-rich regions, that are highly variable in amino acid sequence [36]. For example, two of the best characterized NES sequences are in HIV-Rev (LPPLERLTL) and PKI (LALKLAGLDI) (reviewed in [37]). A rough consensus sequence LX2–3 (F, I, L, V, M)X2–3 LX(L, I) has been formulated [38] but nonetheless, because leucine is a very common amino acid in proteins, NES sequences are difficult to identify with accuracy using database searches. This consensus identified only 36% of experimentally defined leucine-rich NES sequences assembled into a database [39], which can be accessed at: http:// www.cbs.dtu.dk/databases/NESbase/. This group is currently building a prediction method, which should greatly facilitate NES identification.

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1.5.3. Predicting whether a protein shuttles between the nucleus and the cytoplasm Many proteins that shuttle between the nucleus and the cytoplasm have identifiable NLS and NES sequences. By definition, any protein that carries an NES must also contain a signal that first targets it to the nucleus. It is presumed that proteins with an NES are likely to shuttle between the nucleus and the cytoplasm. Some of these proteins continually shuttle between the nucleus and the cytoplasm and others undergo regulated relocalization. A second category of shuttling proteins are those involved in RNA transport (reviewed in [40]). One example is the RNA binding protein hnRNP A1. It contains a unique 38 amino acid recognition sequence that is rich in glycines and aromatic amino acids (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAK PRNQGGY) called M9, that mediates both import and export but does not have similarity to other known NLS or NES sequences (reviewed in [37]). It interacts with a distantly related member of the importin-b family, called transportin. 1.5.4. Identifying the import or export signals of a protein To define an NLS in a candidate protein, it is necessary to functionally characterize the region of the protein with NLS activity and demonstrate that it is both necessary and sufficient for nuclear import. The most common approach begins with a deletion analysis to find the protein domain(s) required for proper intracellular localization. However, caution must be exercised since the amino acids comprising the NLS may not be contiguous. Subsequent tests include: (1) mutating residues within the putative NLS and showing that the protein is no longer nuclear; and (2) fusing the putative NLS to a reporter protein, such as GFP, which equilibrates across the NE, to show that it can direct the accumulation of the reporter in the nucleus. Similar approaches can be used to delineate a functional NES. One approach that can be used to easily test a protein domain for NLS or NES activity in S. pombe is to clone it into the multiple cloning site of plasmid pREP-GFP [33] to express a fusion protein of the putative transport signal and GFP. 1.5.5. Regulation of transport Theoretically, the transport of a protein or class of proteins across the nuclear envelope could be regulated by changes in the transport receptors, the components of the nuclear pore or the interaction between the cargo and the receptor (reviewed in [41,42]). It may also be influenced by the anchoring of cargoes in the cytoplasm or the nucleus, as has been shown for the S. pombe La1 RNA binding protein [43]. For individual proteins, modifications such as phosphorylation, ligand binding or proteolysis have been shown to change the accessi-

bility of the NLS or NES to its receptor thereby altering their intracellular localization (reviewed in [42]). In S. pombe, it has been shown that MCM protein complex formation in the cytoplasm is essential for its nuclear import [33]. Many proteins also have functional NESs, whose activity is regulated by post-translational modifications or protein–protein interactions [44] and LMB is useful for analyzing this type of regulation of nuclear export. 1.6. Investigating nucleocytoplasmic transport in S. pombe 1.6.1. How to determine if a mutant is defective in nuclear protein import or export There is not yet an in vitro system in which to monitor nucleocytoplasmic transport in S. pombe, so only in vivo experimental approaches will be described here. Any mutant defective in the general transport machinery should influence the nucleocytoplasmic transport of all categories of cargo. The choice of reporter cargo substrate is critical, however, when studying mutants defective in specific import receptors, which would be expected to mediate the transport of only a sub-set of transport cargoes. The transport of specific cargoes can be analyzed using immunocytochemistry, and antibodies to many S. pombe proteins have been produced. A simpler approach, that allows the visualization of nucleocytoplasmic transport in living cells, relies on tagging the cargo protein of interest with GFP. 1.6.1.1. Description of method for monitoring nuclear protein import using constitutive expression of a nuclear reporter protein. One method that has been successfully used to study defects in nuclear protein import in S. cerevisiae relies on the fact that an imported protein will accumulate in the cytoplasm when import is blocked. However, when using this approach to study a temperature-sensitive import mutant, the reporter protein will accumulate in the nucleus prior to the temperature shift. The success of this method therefore depends on the continuation of new protein synthesis at the restrictive temperature, so that non-transported protein can accumulate in the cytoplasm at sufficient levels to be visualized. If a reporter is not exclusively nuclear, an import defect would have to be severe enough to be distinguished from a background level of cytoplasmic fluorescence. The NLS of the SV40 large T antigen is commonly used to monitor import in eukaryotic cells because it is competent to direct proteins to which it is fused into the nucleus in animal cells, budding yeast, and fission yeast. Table 1 is a list of some S. pombe GFP-tagged transport reporters that are targeted to the nucleus by exogenous or endogenous NLS sequences.

M. Yoshida, S. Sazer / Methods 33 (2004) 226–238 Table 1 A non-comprehensive list of reagents for analyzing nucleocytoplasmic transport and nuclear envelope structure and integrity in S. pombe Name

Reference

(A) Constitutive nuclear >100 GFP-fusion proteins Pap1p mutants GFP-Hsk1 pREP SV40 NLS-GFP pREP SV40 GFP-MCS pRep GFP pREP SV40 NLS-GFP-LacZa; d pINV1 SV40 NLS-GFP-LacZb Cdt1-CFP Cdc18-CFP, YFP Mcm2-GFP,CFP,YFP Mcm4-GFP,CFP,YFP Mcm6-GFP,CFP,YFP Mcm7-GFP,CFP,YFP Cdc45-YFP Cdc23-CFP Orc6-CFP Tf1 Gag GFP-LacZ nucleoplasmin NLS-GFP

[77] [30] [33] [33] [33] [33] This paper This paper [78] [78] [78] [78] [78] [78] [78] [78] [78] [79] [50]

(B) Shuttling proteins GST-NLS-GFP-NES Mex67-GFP Crp79-GFP Pap1 mutants Srk1-GFP pREP-GFP-Pap1 Byr2-GFP Mei2-GFP Ste11-GFP Cdc25 Spc1/Sty1 Mid1 La1

[17] [63] [59] [30] [72] [46] [73] [28] [29] [27,74] [75] [76] [43]

(C) Nuclear envelope Nup124-GFP Npp106 Nup107-GFP Nsp1GFPc;d

[79] [62] [80] This paper

a The Sazer laboratory constructed plasmid pREP4X-GFP-NLSLacZ, in which expression is controlled by the nmt1 promoter in the ura4þ based plasmid pREP4X [81]. The NLS-GFP-LacZ containing fragment from the S. cerevisiae plasmid pPS817 [82] was subcloned into pREP4X. This plasmid was integrated into the genome of wildtype cells to construct strain SS482 (h-int:GFP-NLS-LacZ/pREP4X:ura4þ leu1-32 ade6-M216 ura4-D18). b The Sazer laboratory constructed plasmid pINV1-GFP-LacZ (SS379) by inserting the gene encoding the SV40 NLS-b-gal fusion protein into plasmid pINV1-GFP [83]. c The Sazer laboratory constructed plasmid pREP82X-GFP-nsp1 by cloning the ORF of nsp1, generated by PCR from genomic DNA, into pREP82X-GFP [81]. This plasmid was integrated into the genome of wild-type cells to construct strain SS715 (h-int:GFP-Nsp1p /pREP82X:ura4þ leu1-32 ade6-M216 ura4-D18). d The Sazer laboratory constructed strain SS818, which has integrated copies of pREP4X-GFP-NLS-LacZ and pREP82X-GFP-nsp1 (h-int:GFP-NLS-LacZ/pREP4X:ura4þ int:GFP-Nsp1p/pREP82X:ura4þ leu1-32 ade6-M216 ura4-D18).

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1.6.1.2. Description of method for monitoring nuclear protein import using regulatable expression of a nuclear reporter protein. Induction of expression of a reporter protein just before creating the experimental conditions under which transport is to be assessed would facilitate studies of nuclear protein import. There are few cloned S. pombe promoters from which expression can be rapidly increased. It takes approximately 18 h to de-repress expression from the most commonly used regulatable promoter, nmt1, which is done by removal of thiamine from the media [45]. Expression of SV40 NLSGFP-LacZ from the regulatable invertase promoter in pINV1-GFP-LacZ (see Table 1) allows induction of expression by changing the carbon source from glucose to sucrose and produces a high level of reporter protein in 1 h. Disappointingly, for reasons that are not understood, this induction does not occur at 36 °C. However, this plasmid may be useful for assaying import defects in cold-sensitive mutants, null mutants, strains in which import is perturbed by overexpression, or other experimental conditions that do not require incubation of cells at 36 °C. Depending on the half-life of the protein of interest, it may also be possible to block and release new protein synthesis with cycloheximide, and then follow the relocalization of the protein from one cellular compartment to another. 1.6.1.3. Description of method for monitoring nuclear protein import and export using constitutive expression of a nucleocytoplasmic shuttling reporter protein. A reporter protein that is ideal for monitoring nucleocytoplasmic transport in S. pombe is Pap1p, which can be rapidly induced to re-localize from the cytoplasm to the nucleus or from the nucleus to the cytoplasm by changing the hydrogen peroxide concentration of the growth media. Pap1p is a transcription factor in the oxidative stress response pathway [46] that shuttles between the nucleus and the cytoplasm. Because its intracellular localization is regulated by the balance between nuclear import and export, Pap1p can be used to monitor defects in both of these processes (see Fig. 2). In wild-type cells, GFPPap1p appears predominantly cytoplasmic at steady state. Upon activation of the stress response, or after exposure to hydrogen peroxide, the nuclear export of Pap1p is inhibited and the protein accumulates in the nucleus [46] (Fig. 3C). In the case of hydrogen peroxide, this is probably due to disulfide formation between a cysteine residue in the Pap1p NES and another cysteine residue outside the NES, that masks the NES from the export receptor Crm1, as reported for Yap1p, a Pap1p homologue in S. cerevisiae [47,48]. Wild-type cells treated with 0.2 mM hydrogen peroxide accumulate GFP-Pap1 in the nucleus within 1 h [46]. In cells treated with 0.003% hydrogen peroxide (0.98 mM), this accumulation occurs within 15 min

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[12,13]. Fifteen minutes after the hydrogen peroxide is washed out, GFP-Pap1p is predominantly cytoplasmic [13]. Some plasmids and strains expressing GFP-tagged shuttling proteins are listed in Table 1. Strains in which the gene of interest is integrated into the genome are preferable because all cells will express the GFP-fusion protein at the same level. The following protocol has been used in the Sazer Laboratory [12,13] and is based on the characterization of Pap1p described by Toone et al. [46]: 1. Grow cells expressing GFP-Pap1 in EMM lacking thiamine to mid-log phase. Thiamine (which is present in YE) will repress expression of GFP-Pap1 from the nmt1 promoter. Cells can be stored on YE plates, but should be pre-cultured in EMM lacking thiamine and then inoculated into a larger volume for analysis. 2. If using a temperature-sensitive mutant, pre-incubate cells at the restrictive temperature for the appropriate length of time. 3. Remove a 10 ml aliquot of the starting culture to a small vial. Observe untreated cells. Mutants defective in nuclear protein export will accumulate GFP-Pap1p in the nucleus prior to hydrogen peroxide addition. 4. Remove a second 10 ml aliquot of the starting culture to a small vial. Add 10 ll of a freshly made 1:10 dilution of 30% hydrogen peroxide solution (Sigma, St. Louis, MO), wait for 15 min, and examine to monitor protein import. 5. Remove a third 10 ml aliquot of the starting culture to a small vial. After 15 min of hydrogen peroxide treatment, wash cells once in EMM without hydrogen peroxide, pre-warmed to the restrictive temperature. Resuspend in pre-warmed EMM and examine 15 min later to monitor protein export. 6. Cells are scored based on the intracellular localization of the GFP signal. The three categories of cells are: (1) those in which nuclear fluorescence is brighter than cytoplasmic; (2) those in which cytoplasmic fluorescence is brighter than nuclear; and (3) those in which the fluorescence is uniform throughout the cell, which depending on the experiment could indicate partial import or partial export. Note. Care should be taken so that the cells are not left under the coverslip too long before observation, because this will induce the stress response and cause GFP-Pap1p to relocate to the nucleus even in wild-type cells untreated with hydrogen peroxide. 7. To more precisely assess the efficiency of transport, import or export can be monitored over time to compare the kinetics of protein relocalization or the time required for complete re-import. The relative GFP fluorescence in the nucleus and the cytoplasm can also be quantified, but in this case it is important to demonstrate that the amount of GFP-tagged protein is equivalent in the samples to be compared.

1.6.1.4. Description of method for monitoring nuclear protein import after release of a poison-induced import block. In S. cerevisiae, inhibition of metabolism blocks nuclear protein import and causes a reversible relocation of nuclear proteins, that are small enough to diffuse through the NPC, into the cytoplasm [49]. Blocking energy metabolism with sodium azide, an inhibitor of cytochrome oxidase, and at the same time blocking glucose metabolism with 2-D -deoxyglucose has a similar effect in S. pombe [50]. After washing out these cell poisons, the ability to re-import these proteins can be examined. The following protocol, developed and used in S. pombe by Dr. Tokio Tani [50], is based on the S. cerevisiae protocol of Goldfarb [49]. 1. Grow cells expressing a nuclear localized NLS-GFP reporter protein to mid-log phase in EMM with 2% glucose. For temperature-sensitive mutants, preincubate cells at the restrictive temperature prior to treatment. 2. Spin down 20 ml of cells, wash once in 1 ml water, pellet again, and resuspend in 1 ml of 10 mM sodium azide, 10 mM of 2-deoxy-o-glucose in glucose free EMM. A control experiment should be performed for each mutant to be tested, to insure that the lack of import is not due to loss of viability. 3. Incubate cells for 45 min at 30 °C (or the restrictive temperature) to allow equilibration of the NLSGFP protein between the nucleus and the cytoplasm. 4. At 4 °C, pellet the cells and wash once in 1 ml water. After washing, the cells can be stored on ice for up to 3 h before assaying. 5. Initiate import by pelleting the cells and resuspending them in 100 ll EMM with glucose pre-warmed to the assay temperature. 6. At each assay time point, remove 2 ll cells and observe. Cells are scored based on the localization of the GFP signal in cells in which the boundary between the nucleus and the cytoplasm can be visualized. Import is scored as cells in which the nucleus is brighter than the cytoplasm and lack of import is scored in cells in which the cytoplasm is brighter than the nucleus. 7. Relative import kinetics can be determined by counting samples every minute and plotting the percent of cells in which import occurs over time. 1.6.1.5. Description of method for determining if the intracellular localization of a protein is dependent on the export receptor Crm1p. Regardless of whether a protein appears to be localized to the nucleus or the cytoplasm, it may actually be shuttling between the two compartments. The localization of shuttling proteins depends on the relative rates of import and export. The shuttling may be continuous or involve regulated import and export under different conditions. One test to determine

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whether a protein that appears cytoplasmic actually shuttles between the nucleus and the cytoplasm is to inhibit export and ask if the protein accumulates in the nucleus. To date, the only characterized nuclear protein export pathway that can be blocked experimentally is the Crm1p-dependent protein export pathway. Crm1p can be inhibited either by a temperature-sensitive mutation [18,19] or by treatment with LMB, a chemical inhibitor of Crm1p [20]. Pap1p has a leucine-rich NES containing important cysteine residues in the C-terminal region. In the presence of LMB or hydrogen peroxide, Pap1p rapidly re-localized to the nucleus, due to the inhibition of Crm1p-dependent nuclear export (see Fig. 3). Some electrophilic compounds such as diethylmaleate directly modify the cysteine residues in the NES, impeding access of Crm1p to the NES [30]. The following protocol for the use of LMB to block nuclear protein export in S. pombe was developed and used by Yoshida and colleagues [17,21,25]. 1. Make a 10 or 100 lg/ml working solution of LMB in ethanol or methanol. Since LMB is an unsaturated fatty acid, it is unstable when exposed to oxygen or UV light. Therefore, it should be stored in the dark, under nitrogen (air in the vial can be easily exchanged for nitrogen by blowing into the vial with a needle), at a temperature under )20 °C for long term storage. LMB is available from Minoru Yoshida (RIKEN, Japan) and Sigma (St. Louis, MO), and will be soon be available from Calbiochem (San Diego, CA). 2. LMB should be added to log phase cultures grown in either EMM or YE to a final concentration of 50 ng/ml. Once added to the medium, LMB has a half-life of 10–30 min, so LMB-containing medium (either liquid or agar plates) should be made just before use. 3. Cells should be treated for at least 30 min before observing in a fluorescence microscope. The rate of the changes in the localization depends on the nuclear import activity of a protein of interest. It is not necessary to wash out the drug prior to observation. 4. For monitoring LMB inhibition of protein export, expression of a GFP-tagged shuttling protein whose export is known to depend on Crm1p, such as GFP-Pap1 or GST-NLS-GFP-NES (GLFE) (see Table 1) should be used as a control. 1.6.2. How to determine if the nuclear localization of a protein is dependent on the RanGTPase system Eight different temperature-sensitive mutations have been identified in the S. pombe RanGEF, Pim1p, all of which are competent for nuclear protein import [13]. Because the nuclear envelope fragments after 4 h at the restrictive temperature, assays monitoring the nuclear localization of a protein of interest should be carried out after a 2 h incubation at the restrictive temperature and

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should include controls to distinguish between transport defects and the lack of nuclear envelope integrity (see Section 1.6). GFP-SV40 NLS-b-Gal, GFP-Nsp1p, or another marker of the nuclear envelope can be used to demonstrate that the NE is intact in cells that mislocalize a particular protein of interest (Table 1). There is also a temperature-sensitive mutant of mog1, but it is not defective in nucleocytoplasmic transport [51]. There are currently no temperature-sensitive mutations in other components of the Ran pathway. Null mutants of pim1 (RanGEF) [4], rna1 (RanGAP) [7,52], spi1 (RanGTPase) [13], sbp1 (RanBP1) [53], and mog1 [51,54] have been constructed and are inviable. A spi1 null strain has been constructed that is kept alive by an integrated copy of wild-type spi1 driven by the high strength nmt1 promoter, but is inviable when the promoter is repressed by the addition of thiamine [13]. A sbp1 null strain has been constructed that is kept alive by expression of wild-type sbp1 driven by the medium strength nmt1 promoter, but is inviable when the promoter is repressed by the addition of thiamine [53]. rna1 null cells in which the wild-type gene driven from the lowest strength nmt1 promoter is repressed are viable, so there is no system in which to conditionally repress rna1 expression to examine the null phenotype [7]. 1.6.3. How to determine if the nuclear localization of a protein is dependent on a specific transport receptor Mutations in nuclear protein import receptors would be expected to affect the localization of a subset of nuclear proteins. One importin-a homologue in fission yeast, Cut15p, can support protein import in an in vitro cell free system [15]. However, the temperature-sensitive cut15 mutant is still capable of importing a GFP protein that carries an NLS. A second importin-a protein, Imp1p [14], has not been characterized. There are 13 importin-b-like proteins in the S. pombe genome [14]. It is anticipated but not yet shown that some of these mediate import in conjunction with importin-a and others mediate the transport of proteins with non-classical NLSs. Only a few attempts have been made to identify the cargo of particular import receptors in any experimental system. No correspondence between cargo function and a specific receptor has been identified. For example, in animal cells four different transport receptors can bind and import three different ribosomal proteins [55]. When the individual importin-bs were deleted in S. cerevisiae, in most cases their cargoes still entered the nucleus, indicating functional overlap in importin function (reviewed in [56]). The best characterized mode of transport regulation is an alteration in the cargo that changes its ability to bind to transport receptors (reviewed in [42]). In animal cells, some of the importin-a and import-b transport receptors have tissue specific expression patterns, but the substrates of these receptors and therefore the

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biological significance of this differential expression remain unknown. In fission yeast, the characterization of importin-bs is at an early stage. The Sal3p importin-b protein in S. pombe, similar to S. cerevisiae Kap121, has recently been characterized [57]. Cdc25p is mislocalized to the cytoplasm in cells carrying a temperature-sensitive mutation in sal3. It remains to be shown that cdc25 is the direct substrate of the Sal3p importin. 1.6.4. How to determine if a mutant is defective in RNA export 1.6.4.1. Description of method for fluorescence in situ hybridization to detect the nuclear accumulation of poly(A)þ RNA. The best available method of monitoring the nuclear export of mRNA is to assay for the retention of poly(A)þ RNA in the nucleus. It should be kept in mind that defects in other aspects of RNA biology, such as splicing or the addition of excessively long poly(A)þ tracks, would also be detected using this method. Fluorescence in situ hybridization has been successfully employed in S. pombe [32,58] to identify mutants defective in mRNA export and the genes for nine of these mutants have been cloned [32,50,58–63]. One of these strains, ptr2 [32], is mutated in pim1, a mutant which had previously been shown to accumulate poly (A)þ RNA in the nucleus at the restrictive temperature [64,65]. S. pombe Los1p, which is the structural homologue of S. cerevisiae Los1p, a protein required for tRNA export [66], is not essential in S. pombe (N. Ong and S. Sazer, unpublished results). The following protocol was developed in the Gould laboratory (personal communication) based on methods described by Forrester [67] and Cole [68] in S. cerevisiae, and Dhar in S. pombe [58]. One significant difference is that in this method the cells are fully processed in liquid whereas in the other methods fixed cells are first adhered to coverslips and then processed. 1. To 5–10 ml of log phase cells, add paraformaldehyde (32% solution, EM grade, Electron Microscopy Sciences, Washington, PA) to a final concentration of 4%. 2. Incubate for 30–60 min in shaking water bath. 3. Wash twice in 0.1 M potassium phosphate, pH 6.5 (K–Pi buffer). 4. Wash once in K–Pi plus 1.2 M sorbitol (K–Pi/SORB). 5. Resuspend in 1 ml K-Pi/SORB. 6. Add 3 ll b-mercaptoethanol and incubate for 10 min. 7. Add 30 ll of 10 mg/ml solution of Zymolyase 20T (ICN Biomedicals, Aurora, OH) and incubate for 20–40 min with rotation until >50% of cells appear spheroplasted. 8. Wash cells three times with K–Pi/SOB, one time in K– Pi. Once in K–Pi + 0.1% NP-40, once in K–Pi. All spins must be done at low speed (3000–4000 rpm).

9. Resuspend in 100 ll pre-hybridization buffer (50% formamide, 4
SSC, 1
DenhardtÕs solution, 125 lg/ml tRNA, 10% dextran sulfate, and 500 mg/ ml denatured salmon sperm DNA). It is best to maintain a stock of pre-hybridization buffer without salmon sperm DNA, which should be denatured and added immediately prior to experimental use. Incubate for 1 h at 37 °C. 10. Add 418 pg/ll oligo(dT)50 probe, 30 -end labeled with digoxigenin-11-dUTP (Roche, Indianapolis, IN), 1.5 U/ll terminal transferase, 20 lg salmon sperm DNA, 20 lg tRNA, 2
SSC, 2% BSA, 10 mM vanadyl ribonucleotide complexes, and 10% dextran sulfate, as previously described [67], and incubate overnight at 37 °C with rotation. 11. Wash cells for 1 h in 2
SSC at room temperature. 12. Wash cells for 1 h in 1
SSC at room temperature. 13. Wash cells for 30 min in 0.5
SSC at 37 °C. 14. Wash cells for 30 min in 0.5
SSC at room temperature. 15. Wash cells for 5 min in PBAL (0.5 g BSA, 0.9 g lysine– HCl (mono) in 50 ml PBS buffer) at room temperature. 16. Block cells for 1 h in PBAL at room temperature. 17. Resuspend in 50 ll PBAL and 1:25 dilution of rabbit polyclonal anti-digoxigenin antibody conjugated to FITC (Roche, Rockford, IL) and incubate for 3– 4 h at room temperature in the dark with rotation. 18. Wash cells 2–3 times in PBAL avoiding exposure to light. Mount the cells in 90% glycerol with 1 mg/ml p-phenylenediamine and 1 lg/ml DAPI, seal the coverslips with nail polish, and store them at 4 °C. 1.7. Investigating nuclear envelope structure and integrity in S. pombe 1.7.1. Description of method for monitoring nuclear envelope structure by visualizing the localization of NPCs The nuclear pore complex is specifically localized to the nuclear envelope in eukaryotic cells. This means that the size and shape of the nucleus can be monitored by following the localization of nuclear pore protein components, which are called nucleoporins. This can be done by following the localization of GFP-nucleoporin fusion proteins in living cells (see Table 1). Alternatively, NPCs can be visualized using monoclonal antibody MAb414, which was raised to and recognizes animal cell nuclear pores but also recognizes a class of nucleoporins in S. cerevisiae [69] and S. pombe [6]. MAb414 is commercially available from Covance Research Products Berkeley, CA. This protocol was developed in the Sazer laboratory [6] based on the S. pombe anti-tubulin immunolocalization method of Hagan and Hyams [70] and the MAb414 protocol used by Wente et al. [69] in S. cerevisiae. 1. Grow cells in minimal media to 2–4
106 /ml. 2. Filter 10 ml of the culture onto a GF/C filter (Whatman: #1821-025).

M. Yoshida, S. Sazer / Methods 33 (2004) 226–238

3. Fix 30 min at room temperature (RT) in 5–10 ml of: 0.1 M K-phosphate, pH 6.5, 10% methanol, 3.7% formaldehyde (Sigma, St. Louis, MO, grade is fine). 4. Remove filter and spin cells down. 5. Wash 3
1 ml in PEM (100 mM Pipes, pH 6.9, 1 mM EGTA, and 1 mM MgSO4 ). Take up in 0.5 ml PEMS (PEM, 1.2 M Sorbitol). 6. Add 10% (by volume) enzyme mix [4 mg Novozyme 234 (InterSpex San Mateo, CA), 2 mg Zymolyase/ml PEMS (Seikagaku America Falmouth, MA)]. Digest 5 min at RT (time of digestion and concentration of enzymes vary from strain to strain and batch to batch of enzyme). 7. Wash 3
with 2 vol. PEMS. 8. Incubate in 1 vol. PEMBAL (PEM, 100 mM lysine hydrochloride, 1% BSA, and 0.1% sodium azide) for at least a 0.5 h at RT. 9. Incubate in 10 ml of undiluted MAb414 (BAbCo, now available from Covance Research Products

10. 11.

12. 13.

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Berkeley, CA) at 4 °C overnight. A 1:10 dilution can also be used with similar results. Wash 3
with 2 vol. PEMBAL. Add secondary antibody in PEMBAL [ImmunoPure goat anti-mouse IgG, fluorescein conjugated; Pierce (Rockford, IL) #31540; 1:100 dilution] and incubate at RT for 1 h in dark. Wash 3
in 2 vol. PEMBAL. Mount on poly-lysine treated coverslips and stain with DAPI by mounting them in 1 lg/ml DAPI, 1 mg/ml p-phenylenediamine in 50% glycerol.

1.7.2. Description of method for monitoring nuclear envelope structure and integrity using the membrane dye DiOC6 DiOC6 is a lipophilic dye that at low concentrations stains the mitochondrial membranes of budding yeast, but at higher concentration stains the ER and nuclear envelope [71]. It can also be used to visualize the nuclear

Fig. 4. GFP-tagged proteins that localize to the nucleus and to the nuclear periphery can be used to monitor nuclear protein import and nuclear envelope integrity. (A) RanGEFts mutant, pim1-d1, expressing SV40 NLS-GFP-b-Gal and GFP-Nsp1 (see Table 1) incubated at the permissive temperature. The proteins are localized exclusively to the nucleus. (B) RanGEFts mutant, pim1-d1, expressing SV40 NLS-GFP-b-Gal and GFP-Nsp1 (see Table 1) incubated at the restrictive temperature. The GFP-b-Gal is dispersed throughout the cell and nuclear fragmentation causes the GFPNsp1p to lose its circular pattern. (C) Cells expressing both SV40 NLS-GFP-b-Gal and GFP-Nsp1 that are defective in protein import would retain the circular GFP-Nsp1p pattern at the nuclear periphery but would not localize SV40 NLS-GFP-b-Gal to the nucleus. (D) Cells expressing GFPNsp1 in which the protein is exclusively localized to the nuclear periphery.

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envelope in living fission yeast cells [6] using a similar protocol. Koning et al. report that DiOC6 should be diluted in ethanol and stored in the dark, and that the staining efficiency varies with cell concentration and between batches of dye. Although treatment with DiOC6 is not toxic to S. pombe, cells exposed to a mercury vapor lamp in a standard fluorescence microscope rapidly lose viability. The dynamics of the nuclear envelope can be monitored in living cells using confocal or deconvolution microscopy in which the exposure of cells to light is minimized. This protocol, used in the Sazer laboratory to visualize both the nuclear envelope and the DNA in living S. pombe cells [6], is based on the S. cerevisiae method that was devised and described in detail by the Wright laboratory [71]. 1. Prepare a log phase culture (approximately 5
106 cells/ml) in EMM or YE. 2. Remove 1 ml of cells. 3. Add 2 ll of a 25 mg/ml solution of Hoechst 33342 (Sigma, St. Louis, MO). 4. Incubate at the growth temperature of the culture for 5 min. 5. Add 10 ll of a 0.5 mg/ml solution of DiOC6 (Molecular Probes, Eugene, OR) in ethanol. 6. Incubate at the growth temperature of the culture for 5 more minutes 7. Observe using fluorescence microscopy. 1.7.3. Description of method for monitoring nuclear envelope integrity using a GFP-tagged nuclear pore protein and a GFP-tagged soluble nuclear protein Nuclear size and shape can also be monitored in cells expressing a GFP-tagged soluble nuclear protein, such as GFP-b-gal (see Table 1). Strains in which the reporter is integrated into the chromosome are best because they insure a uniform level of expression. Any strain containing a soluble GFP-tagged nuclear reporter can also be used to assess nuclear envelope integrity since the protein will lose its exclusive nuclear localization if the envelope is not intact. However, the uniform cellular distribution of a nuclear reporter could also be caused by a defect in nuclear protein import. To overcome this problem, the Sazer laboratory constructed strains that express both NLS-GFP-bGal and GFP-Nsp1p (Figs. 4A–C) from integrated copies of plasmids pREP3X-SV40NLS-GFP-LacZ and pREP82X-GFP-Nsp1 (see Table 1), strains that express only NLS-GFP-b-Gal, and strains that express only GFP-Nsp1 (Fig. 4D). Cells with defects in nuclear protein import would mislocalize NLS-GFP-b-Gal to the cytoplasm, but would retain a circular nuclear envelope visualized by GFP-Nsp1p (Fig. 4C). Cells with defects in nuclear envelope integrity would mislocalize GFP-b-Gal to the cytoplasm and would no longer have a circular nuclear envelope marked with GFP-Nsp1p (Fig. 4B).

2. Concluding remarks Fission yeast is a unique organism in which to investigate nucleocytoplasmic transport and nuclear envelope structure. Although the proteins necessary for nucleocytoplasmic transport in animal cells and budding yeast are conserved in fission yeast, many aspects of their function are apparently different. For example, mutations in the RanGTPase system [3,13] or in the importin-a receptor Cut15p [15], which are essential for nucleocytoplasmic transport in other systems, do not prevent nuclear protein import in fission yeast. In contrast, inhibition of the nuclear protein export receptor Crm1p does block nuclear export of several identified cargo molecules [16,17,26,27,29]. Understanding the nucleocytoplasmic transport system is becoming increasingly important as investigators discover that intracellular protein localization is critical for the regulation of many cellular processes. For reasons that are not understood, mutations in the RanGTPase system result in a fragmentation of the nuclear envelope at mitosis in fission yeast [6] but not in other organisms. Most reagents are now or will soon be available for genome wide analyses of the intracellular localization of proteins in fission yeast, for investigating the mechanism by which specific proteins are localized to the proper cellular compartment and/or re-localized within the cell in response to signaling pathways, and for identifying the proteins required for nuclear division, and for nuclear envelope structure and function.

Acknowledgments S.S. thanks Eric Chang, Susan Forsburg, Elena Hidalgo, Steven Kearsey, Henry Levin, and Richard Maraia for help compiling Table 1; Ravi Dhar, Tokio Tani, Kathy Gould, and Rob Carnahan for help preparing protocols; and Sandi Salus for drawing Fig. 1 and Yoshihiro Torii for Fig. 4. M.Y. thanks Hiroshi Taoka for help with the sections on Crm1 and LMB and Ayako Kamata for preparing Fig. 3. We thank Sandi Salus and Rajesh Nair for comments on the manuscript and Steven Kearsey (University of Oxford) for permission to cite unpublished results. Work in the Sazer laboratory was supported by a grant from the National Institutes of Health (GM49119). Work in the Yoshida laboratory was supported by the CREST Research Project, JST, Japan.

References [1] I.W. Mattaj, L. Englmeier, Annu. Rev. Biochem. 67 (1998) 265– 306. [2] D. Gorlich, U. Kutay, Annu. Rev. Cell. Dev. Biol. 15 (1999) 607– 660.

M. Yoshida, S. Sazer / Methods 33 (2004) 226–238 [3] S.S. Salus, S. Sazer, in: M. Rush, P. DÕEustachio (Eds.), The Small GTPase Ran, Kluwer Academic Publishers, Boston, 2001, pp. 123–144. [4] T. Matsumoto, D. Beach, Cell 66 (1991) 347–360. [5] S. Sazer, P. Nurse, EMBO J. 13 (1994) 606–615. [6] J. Demeter, M. Morphew, S. Sazer, Proc. Natl. Acad. Sci. USA 92 (1995) 1436–1440. [7] A. Matynia, K. Dimitrov, U. Mueller, X. He, S. Sazer, Mol. Cell. Biol. 16 (1996) 6352–6362. [8] S. Sazer, M. Dasso, J. Cell Sci. 113 (2000) 1111–1118. [9] P. Kalab, K. Weis, R. Heald, Science 295 (2002) 2452–2456. [10] D. Gorlich, EMBO J. 17 (1998) 2721–2727. [11] M. Dasso, Cell 104 (2001) 321–324. [12] U. Fleig, S.S. Salus, I. Karig, S. Sazer, J. Cell Biol. 151 (2000) 1101–1112. [13] S.S. Salus, J. Demeter, S. Sazer, Mol. Cell. Biol. 22 (2002) 8491– 8505. [14] V. Wood, R. Gwilliam, M.A. Rajandream, M. Lyne, R. Lyne, A. Stewart, J. Sgouros, N. Peat, J. Hayles, S. Baker, D. Basham, S. Bowman, K. Brooks, D. Brown, S. Brown, T. Chillingworth, C. Churcher, M. Collins, R. Connor, A. Cronin, P. Davis, T. Feltwell, A. Fraser, S. Gentles, A. Goble, N. Hamlin, D. Harris, J. Hidalgo, G. Hodgson, S. Holroyd, T. Hornsby, S. Howarth, E.J. Huckle, S. Hunt, K. Jagels, K. James, L. Jones, M. Jones, S. Leather, S. McDonald, J. McLean, P. Mooney, S. Moule, K. Mungall, L. Murphy, D. Niblett, C. Odell, K. Oliver, S. OÕNeil, D. Pearson, M.A. Quail, E. Rabbinowitsch, K. Rutherford, S. Rutter, D. Saunders, K. Seeger, S. Sharp, J. Skelton, M. Simmonds, R. Squares, S. Squares, K. Stevens, K. Taylor, R.G. Taylor, A. Tivey, S. Walsh, T. Warren, S. Whitehead, J. Woodward, G. Volckaert, R. Aert, J. Robben, B. Grymonprez, I. Weltjens, E. Vanstreels, M. Rieger, M. Schafer, S. Muller-Auer, C. Gabel, M. Fuchs, C. Fritzc, E. Holzer, D. Moestl, H. Hilbert, K. Borzym, I. Langer, A. Beck, H. Lehrach, R. Reinhardt, T.M. Pohl, P. Eger, W. Zimmermann, H. Wedler, R. Wambutt, B. Purnelle, A. Goffeau, E. Cadieu, S. Dreano, S. Gloux, V. Lelaure, S. Mottier, F. Galibert, S.J. Aves, Z. Xiang, C. Hunt, K. Moore, S.M. Hurst, M. Lucas, M. Rochet, C. Gaillardin, V.A. Tallada, A. Garzon, G. Thode, R.R. Daga, L. Cruzado, J. Jimenez, M. Sanchez, F. del Rey, J. Benito, A. Dominguez, J.L. Revuelta, S. Moreno, J. Armstrong, S.L. Forsburg, L. Cerrutti, T. Lowe, W.R. McCombie, I. Paulsen, J. Potashkin, G.V. Shpakovski, D. Ussery, B.G. Barrell, P. Nurse, Nature 415 (2002) 845–848. [15] T. Matsusaka, N. Imamoto, Y. Yoneda, M. Yanagida, Curr. Biol. 8 (1998) 1031–1034. [16] M. Fukuda, S. Asano, T. Nakamura, M. Adachi, M. Yoshida, M. Yanagida, E. Nishida, Nature 390 (1997) 308. [17] N. Kudo, B. Wolff, T. Sekimoto, E.P. Schreiner, Y. Yoneda, M. Yanagida, S. Horinouchi, M. Yoshida, Exp. Cell. Res. 242 (1998) 540–547. [18] Y. Adachi, M. Yanagida, J. Cell, Biology 108 (1989) 1195–1207. [19] K. Kumada, M. Yanagida, T. Toda, Mol. Gen. Genet. 250 (1996) 59–68. [20] M. Yoshida, S. Horinouchi, Ann. N. Y. Acad. Sci. 886 (1999) 23– 36. [21] K. Nishi, M. Yoshida, D. Fujiwara, M. Nishikawa, S. Horinouchi, T. Beppu, J. Biol. Chem. 269 (1994) 6320–6324. [22] B. Wolff, J.-J. Sanglier, Y. Wang, Chem. Biol. 4 (1997) 139–147. [23] M. Fornerod, M. Ohno, M. Yoshida, I.W. Mattaj, Cell 90 (1997) 1051–1060. [24] B. Ossareh-Nazari, F. Bachelerie, C. Dargemont, Science 278 (1997) 141. [25] N. Kudo, N. Matsumori, H. Taoka, D. Fujiwara, E.P. Schreiner, B. Wolff, M. Yoshida, S. Horinouchi, Proc. Natl. Acad. Sci. USA 96 (1999) 9112–9117. [26] N. Kudo, H. Taoka, T. Toda, M. Yoshida, S. Horinouchi, J. Biol. Chem. 274 (1999) 15151–15158.

237

[27] Y. Zeng, H. Piwnica-Worms, Mol. Cell. Biol. 19 (1999) 7410– 7419. [28] M. Sato, S. Shinozaki-Yabana, A. Yamashita, Y. Watanabe, M. Yamamoto, FEBS Lett. 499 (2001) 251–255. [29] J. Qin, W. Kang, B. Leung, M. McLeod, Mol. Cell. Biol. 23 (2003) 3253–3264. [30] E.A. Castillo, J. Ayte, C. Chiva, A. Moldon, M. Carrascal, J. Abian, N. Jones, E. Hidalgo, Mol. Microbiol. 45 (2002) 243– 254. [31] J. Quinn, V.J. Findlay, K. Dawson, J.B. Millar, N. Jones, B.A. Morgan, W.M. Toone, Mol. Biol. Cell 13 (2002) 805–816. [32] A.K. Azad, T. Tani, N. Shiki, S. Tsuneyoshi, S. Urushiyama, Y. Ohshima, Mol. Biol. Cell 8 (1997) 825–841. [33] S.G. Pasion, S.L. Forsburg, Mol. Biol. Cell 10 (1999) 4043–4057. [34] E. Conti, J. Kuriyan, Struct. Fold Des. 8 (2000) 329–338. [35] M. Cokol, R. Nair, B. Rost, EMBO Rep. 1 (2000) 411–415. [36] L. Gerace, Cell 82 (1995) 341–344. [37] D. Gorlich, I.W. Mattaj, Science 271 (1996) 1513–1518. [38] H.P. Bogerd, R.A. Fridell, R.E. Benson, J. Hua, B.R. Cullen, Mol. Cell. Biol. 16 (1996) 4207–4214. [39] T. la Cour, R. Gupta, K. Rapacki, K. Skriver, F.M. Poulsen, S. Brunak, Nucleic Acids Res. 31 (2003) 393–396. [40] W.M. Michael, Trends Cell. Biol. 10 (2000) 46–50. [41] E.A. Nigg, Nature 386 (1997) 779–787. [42] A. Kaffman, E.K. OÕShea, Annu. Rev. Cell. Dev. Biol. 15 (1999) 291–339. [43] R.V. Intine, M. Dundr, T. Misteli, R.J. Maraia, Mol. Cell 9 (2002) 1113–1123. [44] Y. Yashiroda, M. Yoshida, Curr. Med. Chem. 10 (2003) 741– 748. [45] K. Maundrell, J. Biol. Chem. 265 (1990) 10857–10864. [46] W.M. Toone, S. Kuge, M. Samuels, B.A. Morgan, T. Toda, N. Jones, Genes Dev. 12 (1998) 1453–1463. [47] A. Delaunay, A.D. Isnard, M.B. Toledano, EMBO J. 19 (2000) 5157–5166. [48] S. Kuge, M. Arita, A. Murayama, K. Maeta, S. Izawa, Y. Inoue, A. Nomoto, Mol. Cell. Biol. 21 (2001) 6139–6150. [49] N. Shulga, P. Roberts, Z. Gu, L. Spitz, M.M. Tabb, M. Nomura, D.S. Goldfarb, J. Cell Biol. 135 (1996) 329–339. [50] T. Shibuya, S. Tsuneyoshi, A.K. Azad, S. Urushiyama, Y. Ohshima, T. Tani, Genetics 152 (1999) 869–880. [51] K. Tatebayashi, T. Tani, H. Ikeda, Genetics 157 (2001) 1513– 1522. [52] F. Melchior, K. Weber, V. Gerke, Mol. Biol. Cell 4 (1993) 569– 581. [53] X. He, N. Hayashi, N.G. Walcott, Y. Azuma, T.E. Patterson, F.R. Bischoff, T. Nishimoto, S. Sazer, Genetics 148 (1998) 645– 656. [54] F.J. Nicolas, W.J. Moore, C. Zhang, P.R. Clarke, J. Cell Sci. 114 (2001) 3013–3023. [55] S. Jakel, D. Gorlich, EMBO J. 17 (1998) 4491–4502. [56] L.F. Pemberton, G. Blobel, J.S. Rosenblum, Curr. Opin. Cell. Biol. 10 (1998) 392–399. [57] G. Chua, C. Lingner, C. Frazer, P.G. Young, Genetics 162 (2002) 689–703. [58] J.A. Brown, A. Bharathi, A. Ghosh, W. Whalen, E. Fitzgerald, R. Dhar, J. Biol. Chem. 270 (1995) 7411–7419. [59] A.G. Thakurta, W.A. Whalen, J.H. Yoon, A. Bharathi, L. Kozak, C. Whiteford, D.C. Love, J.A. Hanover, R. Dhar, Mol. Biol. Cell 13 (2002) 2571–2584. [60] A.G. Thakurta, J. Ho Yoon, R. Dhar, Yeast 19 (2002) 803–810. [61] L. Kozak, G. Gopal, J.H. Yoon, Z.E. Sauna, S.V. Ambudkar, A.G. Thakurta, R. Dhar, J. Biol. Chem. 277 (2002) 33580–33589. [62] J.H. Yoon, W.A. Whalen, A. Bharathi, R. Shen, R. Dhar, Mol. Cell. Biol. 17 (1997) 7047–7060. [63] J.H. Yoon, D.C. Love, A. Guhathakurta, J.A. Hanover, R. Dhar, Mol. Cell. Biol. 20 (2000) 8767–8782.

238

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[64] T. Kadowaki, D. Goldfarb, L.M. Spitz, A.M. Tartakoff, M. Ohno, EMBO J. 12 (1993) 2929–2937. [65] T. Tani, R.J. Derby, Y. Hiraoka, D.L. Spector, Mol. Biol. Cell 7 (1996) 173–192. [66] D.J. Hurt, S.S. Wang, Y.H. Lin, A.K. Hopper, Mol. Cell. Biol. 7 (1987) 1208–1216. [67] W. Forrester, F. Stutz, M. Rosbash, M. Wickens, Genes Dev. 6 (1992) 1914–1926. [68] C.N. Cole, C.V. Heath, C.A. Hodge, C.M. Hammell, D.C. Amberg, Methods Enzymol. 351 (2002) 568–587. [69] S.R. Wente, M.P. Rout, G. Blobel, J. Cell Biol. 119 (1992) 705– 723. [70] I.M. Hagan, J.S. Hyams, J. Cell Sci. 89 (1988) 343–357. [71] A.J. Koning, P.Y. Lum, J.M. Williams, R. Wright, Cell Motil. Cytoskeleton 25 (1993) 111–128. [72] D.A. Smith, W.M. Toone, D. Chen, J. Bahler, N. Jones, B.A. Morgan, J. Quinn, J. Biol. Chem. 277 (2002) 33411–33421.

[73] F. Ozoe, R. Kurokawa, Y. Kobayashi, H.T. Jeong, K. Tanaka, K. Sen, T. Nakagawa, H. Matsuda, M. Kawamukai, Mol. Cell. Biol. 22 (2002) 7105–7119. [74] A. Lopez-Girona, J. Kanoh, P. Russell, Curr. Biol. 11 (2001) 50–54. [75] F. Gaits, P. Russell, Mol. Biol. Cell 10 (1999) 1395–1407. [76] A. Paoletti, F. Chang, Mol. Biol. Cell 11 (2000) 2757–2773. [77] D.Q. Ding, Y. Tomita, A. Yamamoto, Y. Chikashige, T. Haraguchi, Y. Hiraoka, Genes Cells 5 (2000) 169–190. [78] J. Gregan, K. Lindner, L. Brimage, R. Franklin, M. Namdar, E.A. Hart, S.J. Aves, S.E. Kearsey, Mol. Biol. Cell (2003). [79] D. Balasundaram, M.J. Benedik, M. Morphew, V.D. Dang, H.L. Levin, Mol. Cell. Biol. 19 (1999) 5768–5784. [80] P.T. Tran, L. Marsh, V. Doye, S. Inoue, F. Chang, J. Cell Biol. 153 (2001) 397–411. [81] S.L. Forsburg, Nucleic Acids Res. 21 (1993) 2955–2966. [82] M.S. Lee, M. Henry, P.A. Silver, Genes Dev. 10 (1996) 1233–1246. [83] J.S. Iacovoni, P. Russell, F. Gaits, Gene 232 (1999) 53–58.