CHAPTER

Assessing Regulated Nuclear Transport in Saccharomyces cerevisiae

14

Christopher Ptak, and Richard W. Wozniak Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada

CHAPTER OUTLINE Introduction ............................................................................................................ 312 14.1 Observing Steady-State Localization of Kap Cargo Proteins ...............................314 14.1.1 GFP-Tagged Nuclear Transport Cargos and Reporters..................... 314 14.1.2 Observing Nuclear Transport Cargos and Reporters by Fluorescence Microscopy ................................................................................ 315 14.1.3 Using Plasmid-Based or Genomically Integrated Transport Reporters .................................................................... 316 14.1.4 Preparing Yeast Cells for Fluorescence Microscopy ........................ 317 14.1.5 Quantifying Nucleoplasmic/Cytoplasmic Fluorescence Intensity Ratios .......................................................................... 318 14.2 Perturbing Nuclear Transport...........................................................................319 14.2.1 Conditional Yeast Mutant Strains ................................................. 320 14.2.1.1 Preparing Temperature-Sensitive (ts) Yeast Strains for Fluorescence Microscopy ................................................................. 320 14.2.1.2 Using the MET3 Promoter to Shutoff Gene Expression........ 322 14.2.1.3 Inhibition of Xpo1T539C Using LMB ..................................... 322 14.2.2 Assessing Cell-Cycle Dependence of Nuclear Transport.................. 323 14.2.2.1 Asynchronous Cultures ...................................................... 323 14.2.2.2 Stage-Specific Cell-Cycle Arrest.......................................... 323 14.3 Materials and Reagents...................................................................................327 14.3.1 Plasmids ................................................................................... 327 14.3.2 Strains ...................................................................................... 327 14.3.3 Material/Equipment .................................................................... 327 14.3.4 Reagents, Buffers, and Media...................................................... 327 Acknowledgments ................................................................................................... 328 References ............................................................................................................. 328

Methods in Cell Biology, Volume 122 Copyright © 2014 Elsevier Inc. All rights reserved.

ISSN 0091-679X http://dx.doi.org/10.1016/B978-0-12-417160-2.00014-X

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Abstract Regulated protein transport between the cytoplasm and the nucleoplasm occurs through nuclear pore complexes and is critical to the function of numerous biological pathways. Saccharomyces cerevisiae has been used as a model system to probe the underlying mechanisms of nuclear transport and how they regulate various physiological processes. This has been facilitated, in part, by studies that couple the microscopic observation of a fluorescently tagged transport cargo’s in vivo localization with numerous genetic and biochemical tools available to yeast researchers. Here, we describe some of these methods as they pertain to studies on regulated nuclear transport.

INTRODUCTION Transporting macromolecules between the cytoplasm and nucleus requires passage through nuclear pore complexes (NPCs). NPCs are massive proteinaceous structures embedded within the nuclear envelope (NE) at positions where the inner and outer NE membranes are fused. Structurally, NPCs form from
30 different nucleoporins (Nups) that assemble into several distinct subcomplexes. These subcomplexes are arranged to generate a pore that possesses a central channel through which all materials exchanged between the cytoplasm and nucleus must move (reviewed in Hoelz, Debler, & Blobel, 2011). While metabolites and small proteins may passively diffuse through the NPC, most proteins, mRNAs, and macromolecular complexes (e.g., ribosomes and RNPs) are actively escorted through pores in association with a nuclear transport factor(s) (NTF). NTFs include karyopherins (Kaps) that direct either the nuclear import (importins) or the nuclear export (exportins) of macromolecular cargos, although some Kaps may facilitate both. Most Kaps, also referred to as b-Kaps, bind their cargos directly. An exception is the heterodimeric a/b Kap complex, (Kap60/ Kap95 in yeast), where the Kap-a component binds the cargo and functions as an adapter between the cargo and the b-Kap (reviewed in Aitchison & Rout, 2012). When binding their cargos, Kaps recognize specific amino acid sequence motifs, termed nuclear localization signals (NLSs) or nuclear export signals (NESs), that typically direct cargos from the cytoplasm to the nucleoplasm, or from the nucleoplasm to the cytoplasm, respectively. In some instances, Kaps recognize NLS or NES sequences that conform to a specific consensus (Table 14.1), although for many cargos the transport signal possesses a particular amino acid character rather than a defined consensus. For example, yeast Kap121p and Kap123p recognize NLSs >25 amino acid residues in length that contain regions enriched in basic amino acid residues. Although recognition of a nuclear transport signal is generally linked to a specific Kap, for some cargos multiple Kaps may bind. Thus, there is always the potential for redundancy with respect to the number of Kaps capable of transporting a specific cargo (reviewed in Chook & Su¨el, 2011; Xu et al., 2010).

Introduction

Table 14.1 Nuclear transport signals Yeast karyopherin

Nuclear transport signal

Kap60p/Kap95p Kap60p/Kap95p Kap121p Kap121p

Monopartite classic NLS Bipartite classic NLS Basic NLS Basic NLS

Kap123p

Basic NLS

Kap104p Kap114p

Basic PY-NLS RS domain

Xpo1p

Leucine-rich NES

Amino acid sequence K-(K/R)-X-(K/R)1 (K/R)-(K/R)-X10–12-(K/R)13/5 K-(V/I)-X-K-X1–2-(K/H/R)2 Basic enriched peptide, >25 amino acids1 Basic enriched peptide, >25 amino acids1 (Basic enriched)4–20-(R/K/H)-X2–5-PY1 >40% arginine–serine dipeptide content1 f-X2–3-f-X2–3-f-X-f where f ¼ L, V, I, F, or M3

Amino acid sequences obtained from 1Chook & Su¨el, 2011; 2Kobayashi & Matsuura, 2013; 3 Xu, Framer, & Chook, 2010. X ¼ any amino acid 3/5 refers to 3 out of 5 consecutive amino acids are either a K or an R.

Although Kap/cargo complexes appear to have no intrinsic directionality with respect to how they move through pores, their role in directional transport, as importers or exporters, depends upon the small GTPase Ran (yeast Gsp1p). During import, importin/cargo complexes move through pores by transiently associating/dissociating with multiple Nups, rich in phenylalanine–glycine repeats, that line the central NPC channel. Once through the pore, the importin/cargo complex interacts with nuclear RanGTP resulting in cargo release and the export of the remaining importin/RanGTP complex back to the cytoplasm. Hydrolysis of RanGTP to RanGDP, catalyzed by a cytoplasmic Ran GTPase-activating protein (yeast Rna1), releases the importin and RanGDP is then imported back into nucleus. Once in the nucleus, RanGDP is converted to RanGTP by a Ran guanine nucleotide exchange factor (yeast Prp20p). During export, the exportin/cargo complex forms in the nucleoplasm and is stabilized through its association with RanGTP. This trimeric complex traverses the pore to the cytoplasm where the Ran GTPase-activating protein stimulates Ran hydrolysis of GTP and conversion of RanGTP to RanGDP. This destabilizes the trimeric complex, releasing the cargo and allowing both the exportin and RanGDP to independently reenter the nucleoplasm (reviewed in Cook, Bono, Jinek, & Conti, 2007). Often, cargos will possess both NLSs and NESs, thus their steady-state localization is dependent upon a competition between these two transport events. Which process dominates is often dependent upon extracellular cues and a cell’s physiological state including, for example, nutrient availability, stress conditions, and cellcycle stage. Often these triggers induce posttranslational modifications of the cargo

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that alter the balance between import and export. For example, in yeast cells, grown under conditions of phosphate starvation, the importin Kap121p actively imports the transcription factor Pho4p into the nucleus where it upregulates the expression of phosphate-responsive genes. Upon phosphate addition, however, Pho4p becomes phosphorylated, reducing its affinity for Kap121p, but augmenting its interaction with the exportin Msn5p. Together these events favor export and cytoplasmic accumulation of phosphorylated Pho4p (Kaffman, Rank, O’Neill, Huang, & O’Shea, 1998; Kaffman, Rank, & O’Shea, 1998). In this chapter, we will describe methods used in the analysis of regulated nuclear transport in the yeast Saccharomyces cerevisiae. These experiments center on the use of fluorescence microscopy to probe the in vivo localization of cargos or nuclear transport reporters fused to a fluorescent protein tag. We also detail specific genetic backgrounds and growth conditions used to study how pathways that control progression through the cell cycle regulate nuclear transport.

14.1 OBSERVING STEADY-STATE LOCALIZATION OF KAP CARGO PROTEINS 14.1.1 GFP-tagged nuclear transport cargos and reporters Assaying nuclear transport in vivo has been facilitated by the use of fusion proteins consisting of a nuclear transport signal-containing cargo protein and a fluorescent protein tag. In this chapter, we will focus our discussion on GFP, but other fluorescent proteins such as mCherry offer alternatives. These fusion proteins generally consist of the cargo protein fused at either their carboxy- or amino-terminus to GFP. Strains producing carboxy-terminal GFP fusions of most yeast proteins may be found as part of a library (Huh et al., 2003) that is commercially available (Invitrogen). Alternatively, a targeted cargo may be tagged in a specific genetic background using transformation and homologous recombination. For this purpose, DNA cassettes, constructed to integrate the coding sequence of a fluorescent protein at the carboxy- or amino-terminal ends of a specific ORF, can be synthesized by PCR using plasmids that have been previously described (e.g., Gauss, Trautwein, Sommer, & Spang, 2005; Longtine et al., 1998) and are available through various agencies (e.g., EUROSCARF). Alternatively, transport signals in specific cargoes can be used in isolation to examine transport events that regulate their nuclear import. This approach has the advantage of separating the analysis of a given transport pathway from other transport signals and interactions that may retain or anchor the cargo in the cytoplasm or the nucleus. These experiments employ nuclear transport reporter proteins consisting of either, an NLS–GFP fusion used for nuclear import studies, or an NLS–NES–GFP fusion used for nuclear export studies. The export reporter includes an NLS, along with the NES, to ensure that the fusion enters the nucleus prior to its export (Stade, Ford, Guthrie, & Weis, 1997). Most nuclear transport signals are fused to two or three tandem GFP moieties in order to raise the reporter size to >50 kDa, which is above

14.1 Observing Steady-State Localization of Kap Cargo Proteins

the NPC diffusion limit of
40 kDa. This reduces the contribution of passive diffusion, thus ensuring that reporter localization is predominantly dependent upon facilitated nuclear transport. In addition, these fusions typically employ GFP variants containing the S65T amino acid substitution, (GFPS65T, eGFP, GFPþ) as, relative to GFP, they exhibit stronger fluorescent signals that are not reduced at elevated temperatures, such as 37  C, and are less susceptible to photobleaching (see Ha, Schwarz, Turco, & Beverly, 1996; Sample, Newman, & Zhang, 2009). A caveat of using transport reporters is that the nuclear transport signal of the target is not always straightforward as only some signals conform to a consensus sequence or exhibit enrichment for a specific amino acid(s) (Table 14.1). As a result, signals are often identified through the generation of cargo truncations, each fused to GFP, that narrow down the region and ultimately the amino acid sequence required to produce an expected localization. A second caveat is that while an extracted sequence can function as a transport signal, this may not be the case within the context of the full-length protein from which it was extracted. Thus, complementary analysis may be required, such as the introduction of amino acid substitutions within a cargo’s putative nuclear transport signal and observing its effect on cargo localization. For an example, see Scott, Cairo, Van de Vosse, and Wozniak (2009) where the NES of Mad1p was defined. Beyond their use in defining a cargo’s nuclear transport signal, reporters may also be used to probe Kap function within specific biological pathways. For example, we employed the NLSPho4-GFP3 reporter (Kaffman, Rank, & O’Shea, 1998) to show that general Kap121p-mediated import is inhibited under conditions of spindle assembly checkpoint activation in yeast (Cairo, Ptak, & Wozniak, 2013).

14.1.2 Observing nuclear transport cargos and reporters by fluorescence microscopy Using fluorescence microscopy, the in vivo localization of cargo–GFP, NLS–GFP, and NLS–NES–GFP fusions can be observed. The steady-state localization of these fusions may vary depending upon the cellular levels of the fusion protein, the composition of the signal, and the numbers of nuclear transport signals in the cargo molecule. Those containing solely an import or export signal are predicted to exhibit a predominantly nuclear or cytoplasmic localization. Those cargos containing both signals can present with various ratios of nuclear to cytoplasm signal dictated by the relative strength of the NLS versus the NES (see Fig. 14.1A, for examples of these localizations). For example, a predominantly nuclear signal may reflect either the en masse import of a cargo or the dynamic import and export of the cargo where the import process is favored over export. In addition, changes in the physiological state of a cell may impact the localization of the cargo either by favoring import or export, or through the inhibition of nuclear transport (Fig. 14.1A and C). It is important to keep in mind that steady-state localization of a fusion protein is also influenced by the presence of cargo-binding partners in the cytoplasm or nucleus that could anchor the fusion protein in a compartment. Thus, the localization of a cargo is not necessarily a reflection of the relative strength of import versus export signal.

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FIGURE 14.1 (A) Various cargo protein localization phenotypes, in this case for the reporter NLSPho4-GFP3, are shown. The NLSPho4-GFP3 gene has been integrated into the strain shown. These include predominantly nucleoplasmic (cells grown in YPD) or, under conditions of nuclear import inhibition, predominantly cytoplasmic (cells grown in YPD þ 20 mg/ml nocodazole), or an intermediate localization between both nucleoplasmic and cytoplasmic (cells grown in YPD þ 20 mg/ml nocodazole). The position of a vacuole is indicated by the asterisk. (B) Shown are cells expressing NLSPho4-GFP3 from either a low-copy CEN/ARS plasmid or from a genomic locus (integrated). Note that the cell-to-cell variation of the fluorescent signal, observed when the reporter is expressed from a plasmid, is lost when the reporter gene is expressed from a genomic locus. (C) In the left-hand panels, cells producing either the NLSPho4-GFP3 or NLSSV40-GFP2 reporter are shown. Genes encoding each reporter have been integrated within their respective strains. These cells were treated with nocodazole and imaged by fluorescence microscopy at the times indicated. These images were used to quantify the average nuclear/cytoplasmic fluorescence intensity ratio (nuclear/cytoplasmic ratio) from 50 cells and these values graphed for each time point. Error bars express standard error. Panel C has been reprinted from Cairo et al. (2013) with permission from Elsevier.

14.1.3 Using plasmid-based or genomically integrated transport reporters Coding sequences for cargo–GFP and transport reporters (e.g., NLS–GFP) are often cloned into yeast CEN/ARS-based expression plasmids (e.g., pRS31X and pRS41X family of plasmids; Sikorski & Hieter, 1989). For example, Kaffman, Rank, and O’Shea (1998) cloned the PHO4 promoter and the NLSPho4-GFP3 coding sequence into the pRS316 plasmid. We have used this construct for the generation of other

14.1 Observing Steady-State Localization of Kap Cargo Proteins

plasmid-based reporters (Scott et al., 2009; Scott, Lusk, Dilworth, Aitchison, & Wozniak, 2005) as: the NLSPho4 sequence may be readily replaced with a coding sequence of interest; the GFP3 provides a strong signal and generates fusions above the NPC diffusion limit; the PHO4 promoter leads to sufficient reporter production and ultimately fluorescent signal. However, cells transformed with these “low-copy” plasmids contain one to several copies of the plasmid per cell. Thus, there is often significant cell-to-cell variability in the expression of the fusion gene and levels of the GFP signal strength (Fig. 14.1B). This variability is further exacerbated when high-copy number plasmids are used. This phenomenon can make it difficult to assess fusion protein localization across the cell population. Where necessary, this shortcoming can be overcome by integrating the ORF for GFP at the 30 -end of the cargo gene ORF allowing examination of the cargo–GFP encoded by the gene and regulated by its promoter. Strains producing carboxy-terminal GFP fusions of most yeast proteins may be found as part of a library (Huh et al., 2003) that is commercially available (Invitrogen). Variability in the expression levels of plasmid-based NLS- or NLS–NES–GFP transport reporter genes can also be reduced by their integration. To integrate these reporter genes, we use the plasmids as templates in a PCR to generate a DNA cassette that includes the reporter-coding sequence, relevant regulatory elements (promoter, terminator), and a marker gene. The oligonucleotides used include sequences that base pair with plasmid sequence adjacent to the region that encodes all of these elements. In addition, the oligonucleotides include 60 nucleotides of flanking sequence used for integration of the DNA cassette at a genomic locus. The loci we have used include auxotrophic markers (e.g., ura3 or leu2) within the yeast strain of choice (Cairo et al., 2013). We observe that strains producing these reporters from a genomic locus produce a reporter-GFP signal that is equivalent from cell to cell (Fig. 14.1B).

14.1.4 Preparing yeast cells for fluorescence microscopy The following describes a generalized approach to assess the localization of GFPlabeled fusion proteins from which the underlying nuclear transport process is inferred. Variations of this protocol form the basis of those described in subsequent sections. Equipment and reagents required are listed in Section 14.3. Growth media used will depend upon whether the GFP fusion gene under study is expressed from a plasmid (Synthetic complete (SC) media designed for plasmid selection) or integrated and expressed from a genomic locus (Yeast extract Peptone Dextrose (YPD) media). Note: Adenine supplementation. When working with any ADE3þ ade2-1 strain of S. cerevisiae, (e.g., W303- or YPH-based strains), adenine is added from a 50  stock solution (6 mg/ml adenine in 0.1 N NaOH) to a final concentration of 120 mg/ml. This helps prevent background arising from the accumulation of a fluorescing metabolic intermediate produced as a consequence of the ade2-1 mutation.

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PROTOCOL 1. Inoculate 5 ml of appropriate liquid culture media (YPD or SC) with desired yeast strain and incubate the culture overnight at room temperature (RT) with agitation, for example, on a roller-drum. For larger cultures ( 10 ml), use an appropriately sized erlenmeyer flask and grow the cultures on an orbital shaker. This is required for methods described in subsequent sections where cultures are split or used in a time course. Incubation at RT helps prevent overgrowth, producing an overnight culture at mid to late log phase or OD600 between
0.5 and 2.0. An OD600 of 1.0 is generally equivalent to
2  107 cells/ml. 2. The following day, the culture is diluted to an OD600 of
0.1–0.3, and incubated at desired temperature for two or more generations to an OD600 of
0.5–1.5. We avoid using cultures with too low (2.0) an OD600. 3. Transfer 1 ml of culture to a microcentrifuge tube and collect the cells by centrifugation at
13,000  g for 30 s in a microcentrifuge. Cells grown in SC media tend not to pellet completely. To circumvent this, 100 ml of YPD can be added to the 1 ml of culture prior to centrifugation. This improves the efficiency of cells pelleting. 4. Aspirate off the media and wash the cells with 1 ml of fully supplemented SC media, that is, all amino acids present. As components of YPD will fluoresce, and hence obscure sample observation, this step is particularly useful when YPD is used. 5. Collect the cells by microcentrifugation. Aspirate off the media and resuspend the cells in
20–50 ml of fully supplemented SC media. This should provide a cell density giving between 20 and 100 cells per field when viewed by microscopy at 100  magnification. 6. Spot 1.5 ml of the cell suspension onto a slide. From just above the spot, drop a coverslip onto the cells to promote cell spreading. This should provide an even, single layer of cells that do not move and are observable in the same focal plane. Slides made in this manner should be useable for
15 min before they begin to dry out. 7. We typically visualize cells by epifluorescence microscopy at 100  magnification. Acquisition times will be dependent upon the specific GFP fusion, but should be limited so as not to produce a saturated signal. This is critical for signal quantification as described in the next section.

14.1.5 Quantifying nucleoplasmic/cytoplasmic fluorescence intensity ratios Alterations in transport mediated by specific Kaps, or of targeted cargos, in response to changes in cell physiology often represent an area of keen interest. Changes in the steady-state localization of the cargo–GFP can often be subtle and assessing potential alterations often requires quantifying the relative nuclear/cytoplasmic ratio of cargo– GFP fusions. To do this, we determine the relative amount of GFP fusion

14.2 Perturbing Nuclear Transport

fluorescence observed in the nucleus and cytoplasm of cells. Specifically, Image J software is used to determine the mean integrated fluorescence intensity per unit area in each compartment, which is then expressed as a nuclear/cytoplasmic fluorescence intensity ratio. This analysis is performed as follows. PROTOCOL 1. Acquire images by fluorescence microscopy as described in Section 14.1.4, using various acquisition times. Various exposures yielding subsaturated signal intensity will be used in the following steps to quantify nuclear/cytoplasmic fluorescence intensity ratios. 2. Open a micrograph in Image J and, using the “rectangular” tool, make a 5 pixel  5 pixel box. 3. Place the box within the nucleus of one cell and determine the nuclear mean integrated fluorescence intensity/unit area for the boxed area by clicking the “Measure” tool found under the “Analyze” heading. For GFP fusions possessing an NLS, the nucleus is usually observed as a spot enriched in GFP fluorescence. When the nucleus is not apparent, NPC or NE markers, such as Sec63-mCherry, may be used as a marker for the nuclear periphery (e.g., see Van de Vosse et al., 2013). 4. Repeat step 2 for a cytoplasmic region, as well as a region outside the cell that will define the background signal. When choosing a cytoplasmic region avoid vacuoles as they generally lack fluorescence and will give a false low value for cytoplasmic fluorescence intensity (Fig. 14.1A, asterisk). 5. Subtract the background from both the nuclear and cytoplasmic intensities. 6. Calculate the intensity ratio by dividing the nuclear intensity by the cytoplasmic intensity. 7. Repeat the process for 50 or more cells. Use these values to calculate an average nuclear/cytoplasmic fluorescence intensity ratio. Calculated ratios for a given exposure time should be similar to those determined for other subsaturated exposures. See Fig. 14.1C, for an example.

14.2 PERTURBING NUCLEAR TRANSPORT Cargo localization, as observed using the techniques described above, reflects the steady-state distribution of the cargo as determined by relative rates of import and export under a given growth condition. Understanding the regulation of these transport events requires identifying the factors and physiological conditions that mediate and modulate the cargos localization, that is, what Kaps control the cargo’s localization and what cellular events alter its transport. To probe these mechanistic details, changes in cargo localization within specific mutant backgrounds and/or under specific growth conditions are used. Below, we describe some conditions we have used to assess mechanisms of cargo localization, focusing on the use of conditional yeast mutants and cell-cycle analysis.

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It should be noted that the methods described below are not useful in studying nuclear import processes where either, there is little or no exchange of the cargo between nucleoplasm and cytoplasm after import, or when treating the cells causes a weak transport defect with no observable change in the nuclear signal of an NLS– GFP reporter. The first limitation may be overcome through the use of an inducible system where production of the cargo–GFP is activated only after the cells have been subjected to a specific perturbation, and its import, or lack thereof, observed (see Leslie, Timney, Rout, & Aitchison, 2006). To observe weak import defects, yeast cells, producing a reporter composed of an NLS fused to a single GFP, are treated with metabolic poisons that inhibit facilitated nuclear transport. This allows the NLS–GFP to passively diffuse and equilibrate between cytoplasm and nucleus. Facilitated transport is then restored upon removal of the poisons and the import rate of the reporter determined. By comparing the import rate in treated and untreated cells, the affect of a specific perturbation on nuclear import can be assessed (see Leslie, Timney, Rout, & Aitchison, 2006; Roberts & Goldfarb, 1998).

14.2.1 Conditional yeast mutant strains The use of conditional mutants has proved to be a powerful tool for investigating nuclear transport processes. In yeast, strains carrying a null mutation for nonessential genes are readily made (Tong et al., 2001) and are also available commercially (Invitrogen). Transport in mutants lacking nonessential genes can be directly examined in parallel with a WT counterpart. However, many genes involved in transport and/or its regulation are essential and can only be studied using a conditional mutation. Most of these are temperature-sensitive mutations and include those that are either directly engaged in transport (e.g., kapts, gsp1ts, prp20ts, etc.) or in other biological pathways that directly or indirectly regulate transport, for example, cell-cycle and/or signal transduction pathways. Alternatively, genes of interest may be placed under control of a conditional promoter element, such that they are only expressed under specific growth conditions. An example is use of the MET3 promoter (PMET3) that activates gene expression only when cells are grown in media lacking methionine (Mao, Hu, Liang, & Lu, 2002). A third case is use of a mutant that sensitizes the S. cerevisiae exportin Xpo1p to the antifungal drug Leptomycin B (LMB). While most eukaryotic homologs of Xpo1p are LMB sensitive, the S. cerevisiae homolog lacks a conserved cysteine residue that when alkylated by LMB results in export inhibition. Inserting this cysteine residue (xpo1T539C) renders S. cerevisiae Xpo1p sensitive to LMB (Neville & Rosbash, 1999). Use of these conditional strains is described below.

14.2.1.1 Preparing temperature-sensitive (ts) yeast strains for fluorescence microscopy Note: When using ts yeast strains, fusions should include GFP derivatives carrying the S65T amino acid substitution as GFP itself produces a significantly weaker fluorescent signal at higher temperatures such as 37  C (Sample et al., 2009).

14.2 Perturbing Nuclear Transport

PROTOCOL 1. Follow the protocol described in Section 14.1.4 to establish actively growing cultures of the targeted ts mutant and its WT counterpart. The WT culture is used as a control to ensure that any defects observed in the ts mutant are attributable to a loss of function rather than temperature changes. Cultures are initially grown at a permissive temperature, preferably one that minimizes any defects associated with the mutation. As many mutants isolated based on restricted function at 37  C also show partial defects at 30  C, we tend to grown all cultures at RT prior to shifting to the nonpermissive temperature. 2. Once cultures grown at the permissive temperature have reached the desired cell density, they are split each into two separate cultures. If necessary, dilute the culture to an OD600 of
0.1–0.3 with media at the same temperature to prevent saturation over the course of the temperature shift. Keep one culture of each strain at the permissive temperature and transfer the other culture directly to an air shaker-incubator set at the nonpermissive temperature (37  C for most strains). An air shaker allows for a gradual increase in the temperature of the culture and prevents a heat-shock response that might occur if the cells were transferred directly to a preheated water bath shaker at the nonpermissive temperature. 3. The time of incubation at the nonpermissive temperature, prior to the analysis of transport, is dependent on the mutant. Generally, we examine various time points after temperature shift (between 0 and 3 h) to assess transport changes and, if applicable, their relationship to known phenotypes of the mutant. 4. At the various time points, cells in each culture are processed for microscopy as outlined in Section 14.1.4. Processing and image acquisition are usually done at RT, thus, the time spent to carry out these steps should be quick (
10–15 min maximum) to minimize potential recovery of cells grown at the nonpermissive temperature. If recovery of mutant protein function is rapid, solutions used to prepare the cells can be maintained at the nonpermissive temperature and a heated microscope stage can be used during image acquisition. Note: When many cultures are being analyzed during the same experiment, it is useful to stagger their initial transfer to the nonpermissive temperature, for example, every 15 min and then process in a similar staggered fashion to ensure that each culture is treated in an identical manner. Alternative approaches: Cells sampled at various time points can also be fixed by the direct addition of formaldehyde (Sigma) to a culture to a final concentration of 4%. Cultures are then incubated an additional 5–30 min prior to preparation for microscopy as described in Section 14.1.4. As fixation may inhibit the fluorescence of some GFP fusions, the affect of formaldehyde treatment should be tested prior to its experimental use. For in vivo studies, microfluidic chambers (available from CellASIC Corporation, San Leandro, CA, USA), designed to allow for rapid changes in media and environment (e.g., temperature) using a flow control system, can also be used.

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14.2.1.2 Using the MET3 promoter to shutoff gene expression This method requires replacing the promoter driving a gene of interest with PMET3. We have employed a PCR-based integrative approach described elsewhere, using, as a template, a plasmid that has been modified to include PMET3 (Makio et al., 2009). Methionine auxotrophs may not be used for this technique, as strains are grown in media lacking methionine. The DNA cassette used to introduce PMET3 at a given locus also introduces a selectable marker, which can be used to identify putative positive integrants. Of note, when essential genes are placed under control of the PMET3 promoter, the strain must be grown in media lacking methionine to allow expression of the essential gene. In addition, we generally use a PMET3 cassette containing the coding sequence for an HA3 tag engineered to insert at the amino-terminal end of the target gene open reading frame (ORF). PROTOCOL 1. Inoculate 5 ml of SC media lacking methionine with a colony of the desired yeast strain and incubate the culture overnight with agitation to mid-log phase (OD600
0.5). For most strains, incubate at 30  C. 2. Pellet the cells by centrifugation at
1700  g for 2–3 min using a tabletop swinging bucket centrifuge and then discard the media. 3. Resuspend the pellet in 5 ml of YPD and supplement the culture with 20 mg/ml methionine (5 ml of 20 mg/ml methionine in ddH20). Alternative: If the cultures used must remain in SC media, for example, to select for a plasmid, methionine can be added directly to the culture in step 1 to a final concentration of 200 mg/ml. 4. Incubation time required to deplete the protein encoded by the gene of interest must be determined for each gene product as it is dependent upon the turnover rate of the protein. Preliminary experiments should include a time course that follows depletion of the HA3-tagged protein using Western blot analysis. 5. Process cells in each culture for microscopy as outlined in Section 14.1.4.

14.2.1.3 Inhibition of xpo1T539C using LMB To analyze the function of yeast Xpo1 in the localization of a targeted cargo, the endogenous XPO1 gene can be replaced with the xpo1T539C allele in a genetic background of interest (Neville & Rosbash, 1999). For example, we have defined a putative NES present in the yeast spindle assembly checkpoint protein Mad1p, by engineering the Mad1p NES sequence into a plasmid encoding a NLSSV40-NESMad1pGFP3 reporter and transforming this plasmid into an xpo1T539C-containing strain (Scott et al., 2009). As first demonstrated using an NLSSV40-NESPKI-GFP3 reporter (Stade et al., 1997), an active NES will prevent nuclear accumulation of the reporter. Thus, this reporter can be used to assess the functionality of a putative NES by examining its nuclear exclusion. Moreover, the role of Xpo1 for mediating the export of the NES-containing reporter or cargo can be assessed by treatment of the xpo1T539C strain with LMB and monitoring its nuclear accumulation.

14.2 Perturbing Nuclear Transport

PROTOCOL 1. Cultures of each strain are grown following the protocol outlined in Section 14.1.4 using the appropriate SC media to select for the plasmids if necessary. 2. Once the cells reach an OD600
0.5, split each culture into two. Treat one set with vehicle (20 ml methanol/ml of culture) and the other with LMB to a final concentration of 100 ng/ml (20 ml of 5 mg/ml LMB in methanol per ml of culture). 3. Incubate the cultures at 30  C (for most strains) and examine reporter localization using fluorescence microscopy at 15 min intervals. Generally, for a reporter such as NLSSV40-NESPKI-GFP3, nuclear accumulation is visible in 0.5–1 h.

14.2.2 Assessing cell-cycle dependence of nuclear transport Many nuclear transport processes are regulated in a cell cycle-dependent manner. These may include regulatory events that occur as the cell passes through a particular cell-cycle stage, or may result from cell-cycle arrest in response to activation of a specific checkpoint. Examples include a transport switch for the Sumo E3 ligase Siz1p from principally import (i.e., nuclear accumulation) during G1 and S-phase to export during mitosis (i.e., cytoplasmic localization and septin ring association) ( Johnson & Gupta, 2001; see Fig. 14.2A). In another case, we find that Kap121pmediated nuclear import is inhibited in response to kinetochore/microtubule detachment leading to the loss of nuclear accumulation (Cairo et al., 2013).

14.2.2.1 Asynchronous cultures Possible cell cycle-dependent changes in the localization of a cargo may be assessed in asynchronous cultures. A rough gauge of which cell-cycle stage a cell is currently in employs a visual comparison of mother cell size with the presence and size of the emerging daughter bud, where, unbudded and small budded cells are in G1 phase, small-medium budded cells are in S–G2, and medium–large budded cells are in various stages of mitosis. A finer indicator of cell-cycle stage combines bud size with an additional cell-cycle indicator such as nuclear position and morphology (Hartwell, Culotti, Pringle, & Reid, 1974). The nucleus may be visualized using the cargo if the GFP fusion is imported. Alternatively NPC or NE markers, such as Sec63-mCherry, may be used as a marker for the nuclear periphery. Visualization of Siz1-GFP provides an example of using these cell-cycle characteristics to observe its reduced nuclear localization and appearance in the cytoplasm and at the budneck as cells enter mitosis (Fig. 14.2A).

14.2.2.2 Stage-specific cell-cycle arrest In some cases, it is desirable to arrest cells at a particular cell-cycle stage prior to visualization of the cargo by fluorescence microscopy. There are numerous methods that arrest yeast cells in particular stages of the cell cycle (see Amon, 2002). Here, the

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FIGURE 14.2 (A) Changes in Siz1p localization at various cell-cycle stages are shown. Cells are shown as a progression through the cell cycle and include unbudded (G1), small budded (G1–S), medium budded (S–G2), and large budded (mitosis) cells. The nucleus is apparent as the region showing accumulation of Siz1-GFP signal. Of note, the location and morphology of nuclei in these various cells are indicative of their position in the cell cycle. Cells in G1, S, and G2 phases possess a single nucleus in the mother cell that is positioned away from the budneck. In metaphase, a single nucleus is found at the budneck. During early anaphase, chromosome segregation begins and the nucleus enters the daughter cell. As anaphase progresses, two distinct nuclei form and eventually migrate to the distal ends of the mother and daughter cells by late anaphase/telophase. (B) Shown are FACS profiles for yeast cells from a single culture incubated under various conditions. Initially, the culture was grown asynchronously (AS) to mid-log phase in pH 5.0 YPD. a-Factor was then added and the culture incubated for 3 h prior to sampling for FACS. The cells were then pelleted, washed, and resuspended in YPD containing 20 mg/ml nocodazole and 1% DMSO. Samples for FACS analysis were then taken from this culture every 20 min. FACS profiles shown measure the number of cells (cell count) with a specific DNA content (fluorescence). DNA content is characteristic of specific cell-cycle stages where 1n represents cells with unreplicated DNA in G1, while cells with a DNA content of 2n have replicated DNA and are in G2 or M. a-Factor arrest causes accumulation of cells in START (G1) with a 1n DNA content, while nocodazole treatment arrests cells in M-phase with a 2n DNA content. Cells in S-phase have a DNA content intermediate to these, for example, the small peak at the 40 min time point after release into nocodazole represents cells undergoing replication.

14.2 Perturbing Nuclear Transport

methods we favor are described, including protocols to arrest cells at START, S-phase, or metaphase. Observations made under these various arrest conditions can reveal changes in the localization of cargo molecules arising from modifications of the cargo itself or the nuclear transport machinery. These data can provide important insight into the functions of the nuclear transport machinery in regulating cellular pathways.

14.2.2.2.1 START arrest using a-factor

The mating pheromone a-factor induces a signal transduction pathway within haploid MATa cells that leads to their arrest, prior to cell-cycle commitment, at START. This arrest is characterized by the accumulation of unbudded cells that form a mating projection or “shmoo” (reviewed in Bardwell, 2005). 1. a-factor is solubilized in ddH20 to 1 mg/ml and stored at 20  C as a 200  stock. Strains to be treated with a-factor must have a MATa mating type. 2. Follow the steps in Section 14.1.4 but use YPD or SC media whose pH has been adjusted to 5.0. The efficacy of a-factor in mediating arrest is much higher at this pH as the Bar1p protease, that degrades a-factor, is inhibited under this condition (Futcher, 1999). 3. Once cells have reached an OD600 no > 0.5, add a-factor to a final concentration of 5 mg/ml and incubate
2.5–3 h. The extent of arrest can be visually determined by microscopy using a brightfield microscope. Efficient arrest generally leads to >90% of cells appearing unbudded and exhibiting a schmooing phenotype (Amon, 2002). 4. Alternative: To reduce the amount of a-factor required for cell arrest, the BAR1 gene can be deleted in the strain being used. Loss of Bar1p hypersensitizes cells to a-factor such that only 10–50 ng/ml are required to induce arrest (MacKay et al., 1988). This also eliminates the need for using pH 5.0 media. 5. Process cells in each culture for microscopy as outlined in Section 14.1.4. For these and subsequent cell-cycle arrest protocols, it is important to confirm the efficiency of arrest using FACS analysis (e.g., see Fig. 14.2B). Cells to be analyzed by FACS are pelleted from 1 ml of mid-log phase culture (
1  107 cells) then fixed by resuspension in 1 ml of 70% ethanol. Prior to further preparation, cells are incubated at RT for 1 h or can be left at 4  C overnight to several days prior to further treatment. Cells are then treated sequentially with RNase, protease, and a fluorescing DNA dye (propidium iodide or SYTOX green) prior to determination of the cells DNA content by flow cytometry (for details, see Hasse & Reed, 2002). 6. Release from arrest: After step 3, cells can be released from arrest, to reenter the cell cycle, by washing the cells twice with media lacking a-factor. After the second wash, the cells are resuspended in the same media and incubated at 30  C. By observing a sample of the culture every 10–20 min by fluorescence microscopy, progression of the cell population, and localization of the transport cargo, through the cell cycle can be followed. We generally do not sample beyond
3 h postrelease as the cultures become asynchronous.

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14.2.2.2.2 S-phase arrest using hydroxyurea 1. Follow the steps in Section 14.1.4; however, on day 2, grow the cultures to an OD600 no greater than 0.5. 2. Add hyroxyurea powder directly to the culture for a final concentration of 250 mM. 3. Incubate for 3 h (approximately two generations). Cells should appear as large budded. These cells generally have a rounder appearance then large budded cells observed in asynchronous populations and also have a single nucleus. 4. Process cells in each culture for microscopy as outlined in Section 14.1.4.

14.2.2.2.3 Metaphase arrest A metaphase arrest can be induced by activation of the spindle assembly checkpoint. Activation of this checkpoint occurs in response to defects in kinetochore– microtubule attachments. Drugs that cause microtubule depolymerization, such as nocodazole, can be used to disrupt these attachments (protocol detailed below). Alternatively, ts mutations in kinetochore components, for example, ndc80ts or ask1ts alleles, sufficiently destabilize the kinetochore–microtubule interface to activate the spindle assembly checkpoint (Pinsky, Kung, Shokat, & Biggins, 2006). The protocol outlined in Section 14.2.1.1 to evaluate cargos in a ts mutant can be employed for kinetochore mutants producing a GFP–cargo. Alternatively, metaphase arrest can be achieved without disrupting kinetochore– microtubule interactions by altering levels of key mitotic regulates such as Cdc20. During mitosis, increasing levels of Cdc20 bind to and activate the anaphase promoting complex. By downregulating the expression of CDC20 using the regulatable MET3 promoter, cells can be arrested in metaphase (Cairo et al., 2013). Using a PMET3-CDC20 strain producing a GFP–cargo, the protocol outlined in Section 14.2.1.2 can be followed to deplete cells of Cdc20p and examine transport in metaphase arrested cells. Moreover, we have used PMET3-CDC20-mediated metaphase arrest to distinguish between transport regulatory events controlled by metaphase arrest (detected in Cdc20 depleted cells) from those activated by disruption of kinetochore–microtubules interactions (detected in nocodazole arrested cells or Cdc20 depleted cells treated with nocodazole). PROTOCOL: NOCODAZOLE TREATMENT 1. Follow the steps in Section 14.1.4; however, on day 2, grow the cultures to an OD600 no greater then 0.3, then split each culture into two. 2. To one of the split cultures, add DMSO to a final concentration of 1% v/v (vehicle only). The other half of the culture is treated with nocodazole as follows. To 1 ml of culture media, add 10 ml of a 200 mg/ml solution of nocodazole in DMSO. The latter solution is made fresh by diluting a nocodazole stock (2 mg/ml in DMSO) 10-fold with DMSO. Nocodazole can also be used at 12.5 and 15 mg/ml concentrations; however, 20 mg/ml works best in our hands.

14.3 Materials and Reagents

In addition, we have found that the efficacy of nocodazole is not equivalent from all vendors. We have had the best success with that obtained from EMD Millipore. 3. Incubate the cells for 2–3 h. We generally find that the majority of cells are arrested and appear large budded by 2–2.5 h. By 3 h and longer, a significant proportion of the population begins to rebud, indicative of slippage out of the metaphase arrest. 4. Process cells in each culture for microscopy as outlined in Section 14.1.4. We generally image at 0.5 h time points starting 1.5 h after nocodazole addition.

14.3 MATERIALS AND REAGENTS 14.3.1 Plasmids pRS31X and pRS41X family of plasmids; (Sikorski & Hieter, 1989) pNLSPho4-GFP3 (pRS316 based; Kaffman, Rank, & O’Shea, 1998) pTM1046 (contains PMET3 that replaces PGAL1 of pFA6a-kanMX6-PGAL1-3HA) (Longtine et al., 1998; Makio et al., 2009)

14.3.2 Strains Yeast deletion library strains (available from Invitrogen) ask1ts strain (W303; Pinsky et al., 2006) bar1△ (BY4741; our lab) ndc80ts strain (W303; Pinsky et al., 2006) PMET3-CDC20 strain (W303; our lab) SEC63-mCherry-NAT (BY4741; our lab) xpo1T539C (W303; Neville & Rosbash, 1999)

14.3.3 Material/equipment Erlenmeyer flasks, orbital shaker, test tubes (16  150-mm tubes for cultures of 5 ml or less), roller-drum, incubator, 1.5 ml polypropylene microcentrifuge tubes, microcentrifuge, glass microscope slides, glass coverslips, fluorescence microscope, flow cytometer (we used a FACScan, Becton Dickinson).

14.3.4 Reagents, buffers, and media Yeast media – 20% Dextrose (per 500 ml): Solubilize 100 g dextrose in double deionized water (ddH2O) to a final volume of 500 ml, and then autoclave. – SC liquid media (per l): 1.7 g yeast nitrogen base, 5 g ammonium sulphate, and 0.8 g amino acid dropout powder (specific for plasmid auxotrophic marker used, ref if commercial, or briefly mention prepared by mixing equivalent amounts of all

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aa þ ade þ ura at concentration XX each?– company is Sunrise Science Products) are solubilized in ddH2O to a final volume of 900 ml and then autoclaved. Once autoclaved, 100 ml of sterilized 20% (w/v) dextrose is added to a final concentration of 2% (w/v). – YPD liquid media (per l): 10 g yeast extract and 20 g peptone are solubilized in ddH2O to a final volume of 900 ml and then autoclaved. Once autoclaved, 100 ml of sterilized 20% (w/v) of dextrose is added to a final concentration of 2% (w/v). a-factor (acetate salt; Sigma): Stock solution: 1 mg/ml in ddH2O. Store aliquots at 20  C Formaldehyde (36.5–38% solution; Sigma) LMB (Sigma): Stock solution: 5 mg/ml in 100% methanol. Store aliquots at 20  C. Hydroxyurea (Sigma): Store powder at 4  C. Nocodazole (EMD Millipore): Stock solution: 2 mg/ml in DMSO.

Acknowledgments Funding sources for this work were provided by the Howard Hughes Medical Institute, Alberta Innovates Health Solutions, and the Canadian Institutes of Health Research (MOP 106502).

References Aitchison, J. D., & Rout, M. P. (2012). The yeast nuclear pore complex and transport through it. Genetics, 190(3), 855–883. Amon, A. (2002). Synchronization procedures. Methods in Enzymology, 351, 457–467. Bardwell, L. (2005). A walk-through of the yeast mating pheromone response pathway. Peptides, 26(2), 339–350. Cairo, L. V., Ptak, C., & Wozniak, R. W. (2013). Mitosis-specific regulation of nuclear transport by the spindle assembly checkpoint protein Mad1p. Molecular Cell, 49(1), 109–120. Chook, Y. M., & Su¨el, K. E. (2011). Nuclear import by karyopherin-bs: Recognition and inhibition. Biochimica et Biophysica Acta, 1813(9), 1593–1606. Cook, A., Bono, F., Jinek, M., & Conti, E. (2007). Structural biology of nucleocytoplasmic transport. Annual Review of Biochemistry, 76, 647–671. Futcher, B. (1999). Cell cycle synchronization. Methods in Cell Science, 21(2–3), 79–86. Gauss, R., Trautwein, M., Sommer, T., & Spang, A. (2005). New modules for the repeated internal and N-terminal epitope tagging of genes in Saccharomyces cerevisiae. Yeast, 22(1), 1–12. Ha, D. S., Schwarz, J. K., Turco, S. J., & Beverly, S. M. (1996). Use of the green fluorescent protein as a marker in transfected Leishmania. Molecular and Biochemical Parasitology, 77(1), 57–64. Hartwell, L. H., Culotti, J., Pringle, J. R., & Reid, J. T. (1974). Genetic control of the cell division cycle in yeast. Science, 183(4120), 46–51.

References

Hasse, S. B., & Reed, S. I. (2002). Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle, 1(2), 132–136. Hoelz, A., Debler, E. W., & Blobel, G. (2011). The structure of the nuclear pore complex. Annual Review of Biochemistry, 80, 613–643. Huh, W.-K., Falvo, J. V., Gerke, L. C., Carroll, A. S., Howson, R. W., Weissman, J. S., et al. (2003). Global analysis of protein localization in budding yeast. Nature, 425(6959), 686–691. Johnson, E. S., & Gupta, A. A. (2001). An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell, 106(6), 735–744. Kaffman, A., Rank, N. M., O’Neill, E. M., Huang, L. S., & O’Shea, E. K. (1998). The receptor Msn5 exports the phosphorylated transcription factor Pho4 out of the nucleus. Nature, 396(6710), 482–486. Kaffman, A., Rank, N. M., & O’Shea, E. K. (1998). Phosphorylation regulates association of the transcription factor Pho4 with its import receptor Pse1/Kap121. Genes and Development, 12(17), 2673–2683. Kobayashi, J., & Matsuura, Y. (2013). Structural basis for cell-cycle-dependent nuclear import mediated by the karyopherin Kap121p. Journal of Molecular Biology, 425(11), 1852–1868. Leslie, D. M., Timney, B., Rout, M. P., & Aitchison, J. D. (2006). Studying nuclear protein import in yeast. Methods, 39(4), 291–308. Longtine, M. S., McKenzie, A., 3rd., Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., et al. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast, 14(10), 953–961. MacKay, V. L., Welch, S. K., Insley, M. Y., Manney, T. R., Holly, J., Saari, G. C., et al. (1988). The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin. Proceedings of the National Academy of Sciences of the United States of America, 85(1), 55–59. Makio, T., Stanton, L. H., Lin, C.-C., Goldfarb, D. S., Weis, K., & Wozniak, R. W. (2009). The nucleoporins Nup170p and Nup157p are essential for nuclear pore complex assembly. Journal of Cell Biology, 185(3), 459–473. Mao, X., Hu, Y., Liang, C., & Lu, C. (2002). Met3 promoter: A tightly regulated promoter and its application in construction of conditional lethal strain. Current Microbiology, 45(1), 37–40. Neville, M., & Rosbash, M. (1999). The NES-Crm1 export pathway is not a major mRNA export route in Saccharomyces cerevisiae. The EMBO Journal, 18(13), 3746–3756. Pinsky, B. A., Kung, C., Shokat, K. M., & Biggins, S. (2006). The Ilp1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores. Nature Cell Biology, 8(1), 78–83. Roberts, P. M., & Goldfarb, D. S. (1998). In vivo nuclear transport kinetics in Saccharomyces cerevisiae. Methods in Cell Biology, 53, 545–557. Sample, V., Newman, R. H., & Zhang, J. (2009). The structure and function of fluorescent proteins. Chemical Society Reviews, 38(10), 2852–2864. Scott, R. J., Cairo, L. V., Van de Vosse, D. W., & Wozniak, R. W. (2009). The nuclear export factor Xpo1p targets Mad1p to kinetochores in yeast. Journal of Cell Biology, 184(1), 21–29. Scott, R. J., Lusk, C. P., Dilworth, D. J., Aitchison, J. D., & Wozniak, R. W. (2005). Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae. Molecular Biology of the Cell, 16(9), 4362–4374.

329

330

CHAPTER 14 Nuclear Transport in Saccharomyces cerevisiae

Sikorski, R. S., & Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics, 122(1), 19–22. Stade, K., Ford, C. S., Guthrie, C., & Weis, K. (1997). Exportin 1 (Crm1p) is an essential nuclear export factor. Cell, 90(6), 1041–1050. Tong, A. H., Evangelista, M., Parsons, A. B., Xu, H., Bader, G. D., Page´, N., et al. (2001). Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science, 294(5550), 2364–2368. Van de Vosse, D. W., Wan, Y., Lapetina, D. L., Chen, W.-M., Chiang, J.-H., Aitchison, J. D., et al. (2013). A role for the nucleoporin Nup170p in chromatin structure and gene silencing. Cell, 152(5), 969–983. Xu, D., Framer, A., & Chook, Y. H. (2010). Recognition of nuclear targeting signals by karyopherin-b proteins. Current Opinion in Structural Biology, 20(6), 782–790.