GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe, we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP–mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP–mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP–mCherry and efficient targeting of Dma1– or Cdc7–GBP–mCherry to the spindle pole body by Sid4–GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale.
A key challenge in cell biology is to directly link protein localization to its function. One way to study the function of a certain protein at a specific site in the cell is to artificially enhance or drive away their localization at that site, followed by functional analysis and characterization. Currently, the most commonly used approach to target proteins to a specific site in the cell is to fuse a protein of interest to another protein that is capable of bringing the fusion protein to the desired cellular site. However, it does not always work as desired because the fusion proteins mostly result in a significant increase in size and interfere with function of the protein of interest.
As one alternative approach to sequester or manipulate protein localization, the anchor-away (AA) system was first developed in the budding yeast Saccharomyces cerevisiae and later adapted to the fission yeast Schizosaccharomyces pombe to deplete nuclear proteins (Ding et al., 2014; Haruki et al., 2008). This system takes advantage of the rapamycin-driven interaction between the FRB- and FKBP12-binding domains of Tor proteins to relocalize nuclear proteins of interest to the cytoplasm (Haruki et al., 2008). Although the AA system allows temporal control and reversibility of binding between the target FRB and anchor FKBP12 constructs, it has a few limitations. First, so far it works only for manipulating some nuclear proteins. Second, the FRB and FKBP12 tags are somewhat bulky and may interfere with functions of the target or anchor proteins. Third, the AA system requires the use of rapamycin-resistant yeast strains of TOR-pathway mutants, which may cause sterility or growth defects.
A 13-kDa soluble protein derived from a llama heavy chain antibody (called GFP-binding protein or GBP) has been recently developed for protein targeting in vivo (Rothbauer et al., 2006, 2008) and rapidly applied to cultured mammalian cells and various model organisms, including fly, worm, Nicotiana, Arabidopsis and S. pombe (Deng and Moseley, 2013; Dodgson et al., 2013; Grallert et al., 2013; Maier et al., 2013; Neumuller et al., 2012; Qu et al., 2013; Rothbauer et al., 2008; Schornack et al., 2009; Sonneville et al., 2012; Tao et al., 2014; Ye et al., 2012). This single-domain antibody features has high binding affinity to GFP as well as to some GFP variants, such as yellow fluorescent protein (YFP), with a stoichiometric ratio of 1:1, but not to cyan fluorescent protein (CFP) or any derivatives of DsRed, such as mRFP, mCherry or mOrange (Kirchhofer et al., 2010; Kubala et al., 2010; Rothbauer et al., 2008). Thus, the GBP can be used to both efficiently purify endogenous protein complexes with GFP tags and target GBP-fusion proteins to different subcellular compartments and structures in live cells through GFP-tagged proteins that have specific localization patterns. The GBP-targeting technology therefore represents a potentially very efficient alternative technique for linking cellular localization to function and complements current methods based on fusion to heterologous localization proteins or signal peptides.
Recent advances of genetics, genomics and molecular biology of the fission yeast S. pombe have rendered it as a powerful model organism to study many aspects of cellular activities. In the past decade, many S. pombe proteins have been individually or systematically tagged with GFP or GFP variants for investigation of localization or for other specific purposes (for example, see Matsuyama et al., 2006). This provides a large available pool of GFP-fusion proteins that can be easily employed to recruit proteins of interest fused with GBP; it also allows targeted manipulation of cellular structures and processes in living fission yeast cells.
We describe here the construction of a series of pFA6a-, pJK148- or pUC119-based vectors (Bahler et al., 1998; Kawashima et al., 2007; Keeney and Boeke, 1994; Tada et al., 2011; Yokobayashi and Watanabe, 2005) containing the GBP or GBP–mCherry fragment and variants of inducible nmt1 or constitutive adh1 promoters with different strengths of gene expression (i.e. Pnmt1, Pnmt41 and Pnmt81 and Padh1, Padh11, Padh21 and Padh81) (Basi et al., 1993; Maundrell, 1990; Kawashima et al., 2007; Tada et al., 2011; Yokobayashi and Watanabe, 2005) that were designed for expression of GBP-fusion proteins in fission yeast. GBP or GBP–mCherry in pFA6a-based vectors serve as cassettes for C-terminal epitope tags, which can be PCR amplified and genomically integrated behind any desired gene and under the control of the native gene promoter. For pJK148- or pUC119-based vectors, the coding sequence of the gene of interest needs to be first cloned at the N- or C-terminus of the GBP or GBP–mCherry fragment, followed by subsequent genomic integration at leu1+ or lys1+ loci. The GBP–mCherry fusion enables easier monitoring of the localization of target proteins upon integration and expression. The use of nmt1 or adh1 promoters with differing strengths of gene expression contained in these vectors should provide flexible choices for control and manipulation over a wide-range of expression levels. To test the feasibility and efficiency of these GBP-targeting vectors, yeast strains with Dma1 (Guertin et al., 2002; Murone and Simanis, 1996) or Cdc7 (Fankhauser and Simanis, 1994) tagged with GBP–mCherry were crossed to strains carrying GFP-fusion proteins localizing at different subcellular compartments and structures. Targeting of these proteins to spindle pole bodies (SPBs) through Sid4–GFP causes abnormal septation phenotypes – such as septation failure (for Dma1) or premature septation (for Cdc7), which are consistent with previous studies based on overexpression strategies (Guertin et al., 2002; Sohrmann et al., 1998) – demonstrating the high efficiency of the GBP-fusion approach. Thus, the series of vectors generated in this study should greatly allow for easier manipulation of protein localization and facilitate studies on gene function in fission yeast.
Construction of three series of vectors for tagging a protein of interest with GBP or GBP–mCherry at the endogenous locus or at leu1+ and lys1+ after targeted integration
By using the ‘T-type’ enzyme-free cloning procedures or combined ‘T-type’ cloning and the conventional restriction digestion-based cloning method, we constructed three series of vectors, which can be used to fuse a protein of interest with GBP or GBP–mCherry at endogenous locus or at leu1+ and lys1+ after targeted integration (Fig. 1A–C; Table S1). Tagging target proteins with GBP–mCherry should allow easy visualization and monitoring of how well the target proteins are anchored by GFP-fusion proteins in yeast cells.
In the first series of vectors constructed (Fig. 1A), GBP alone or GBP–mCherry fusion was assembled into the pFA6a backbone (Bahler et al., 1998), which is commonly and widely used for PCR-based gene targeting in fission yeast with high efficiency. Since the flanking sequences on both sides of GBP or GBP–mCherry were derived from original pFA6a-MX6 plasmids and remained intact, primers designed for gene C-terminal tagging with other tags could be used directly without the need to design new primers. Successful tagging of the gene of interest could be checked with PCR using two primers, in which one primer corresponds to the specific sequence of the gene being tagged and the other universal reverse primer anneals with the upstream region of the drug-resistant cassette (for example, PTEF), or kanMX6 coding region (for example, primer #281, Table S3). The resultant tagged genes are expressed at their endogenous levels since they are driven by their own promoters.
In the second series of vectors constructed (Fig. 1B, Fig. 2), GBP or the GBP–mCherry fusion was first subcloned into a pREP41-EGFP-based vector (Craven et al., 1998), then the GBP and GBP–mCherry fragments flanked by the nmt41 promoter (Pnmt41) and nmt1 terminator (Tnmt) sequences were cloned into the integration vector pJK148 (Keeney and Boeke, 1994). The range of promoter strength was further extended by mutating the TATA box sequences of the nmt1 promoter, giving a series of the nmt1 promoter variants (Pnmt81, Pnmt41 or Pnmt1). The transcription level from these nmt1 promoters can be controlled by addition or removal of thiamine in the medium. The expression is repressed in the presence of thiamine, whereas it is induced in its absence. In addition to carrying these inducible promoters (Pnmt81, Pnmt41 or Pnmt1), which are widely used as artificial promoters to control expression of external genes (Basi et al., 1993; Maundrell, 1990), these ‘mosaic’ plasmids also carry a commonly used target module of leu1+ (Keeney and Boeke, 1994). After being linearized in the leu1+ sequence through digestion with properly chosen unique restriction enzymes (e.g. Bsu36I or NruI), these integration plasmids can be efficiently targeted to their homologous sequences at the genomic locus of the auxotrophic marker leu1-32 on chromosome II by recombination. Since C-terminal tagging does not always yield functional fusion proteins, we also constructed pJK148-based vectors for N-terminal GBP or GBP–mCherry tagging by leaving multi-cloning sites between GBP or GBP–mCherry and nmt1 terminators (Fig. 1B, Fig. 2). This should greatly enhance the chance and flexibility of this series of vectors in successful tagging of any gene of interest.
In recent years, a series of the adh1 promoter variants (e.g. Padh1, Padh11, Padh21 and Padh81) has been developed, and in the meantime, the truncated sequence corresponding to lys1+ gene on chromosome I has been successfully used as a targeting module (Kawashima et al., 2007; Tada et al., 2011; Yokobayashi and Watanabe, 2005). The adh1 promoter (Padh1) is originally derived from the promoter region of the endogenous adh1+ gene, whereas Padh11, Padh21 and Padh81 are mutant versions of Padh1 with weaker promoter activities (Kawashima et al., 2007; Tada et al., 2011; Yokobayashi and Watanabe, 2005). This series of the adh1 promoter variants allows constitutive expression with a wide range of gene expression strengths, which further increase flexibility. The lys1+ gene is a relatively large gene with the total length of the open reading frame being 4260 bp, whereas the truncated version of lys1 used as a target module only carries the middle part of the gene, with a sequence of ∼1.5 kb. We named this truncated version of lys1 as lys1* for convenience. By taking advantage of these tools, we constructed the third series of vectors for GBP or GBP–mCherry tagging by assembling adh1 promoter variants, GBP or GBP–mCherry tags, antibiotic resistance marker hphMX6 and the truncated lys1 gene sequence in the pUC119 backbone (Fig. 1C, Fig. 2). Similar to pJK148-based tagging vectors, we also constructed pUC119-based vectors both for C-terminal and N-terminal GBP or GBP–mCherry tagging to increase the chances of obtaining functional fusion proteins (Fig. 1C, Fig. 2). The integration of these pUC119-based tagging vectors at the genomic lys1+ locus can be achieved after linearization through digestion with properly chosen unique restriction enzymes (e.g. ApaI) within lys1* followed by transformation into yeast cells. It is noteworthy that when pUC119-based tagging vectors are used, correct transformants should confer both antibiotic resistance due to the presence of the hphMX6 cassette (Fig. 1C) and lysine auxotrophy. Lysine auxotrophy is caused by loss of function of lys1+, because correct integration of the DNA fragment at lys1+ locus should disrupt endogenous lys1+ and leave two partially deleted lys1+ gene sequences on either side of the tagged genes, one of which can be called lys1Δ, and the other, lys1*, is introduced by the vectors (see diagram later in Fig. 3A). Therefore, transformants can be tested for with YE5S medium containing hygromycin B and with minimal medium that lacks lysine, and alternatively, they can also be verified using the colony PCR method (see Materials and Methods).
Comparison of protein expression of GBP–mCherry after integration at leu1+ and lys1+ under the control of nmt1 and adh1 promoter variants, respectively, using luciferase as a reporter
We incorporated seven promoters (including three regulatable nmt1 and four constitutive adh1 promoters) into our constructed pJK148- and pUC119-based vectors for GBP or GBP–mCherry tagging and genomic integration. So far, among these commonly used or newly developed promoters in fission, the expression strength of each promoter in yeast studies has been analyzed and compared only for nmt1-variant promoters (Pnmt1, Pnmt41, Pnmt81) and the adh1 promoter (Padh1), using either the chloramphenicol acetyltransferase (CAT)-encoding gene or β-galactosidase-encoding gene (lacZ) as reporters (Basi et al., 1993; Forsburg, 1993). In order to better assess the relative strengths of all adh1 and nmt1 promoter variants, and the abundance of GBP or GBP–mCherry-tagged proteins, we cloned the firefly luciferase (F.luc)-coding sequence into multi-cloning sites of pJK148- and pUC119-based vectors behind the GBP–mCherry tag (Fig. 3A). Thus, the luciferase activity can be measured after these integrated GBP–mCherry–F.luc fusions are expressed in yeast cells, driven by all different adh1 and nmt1 promoter variants, the firefly luciferase therefore serves as a reporter for the abundance of GBP–mCherry fusion proteins. As an internal control, we also tagged endogenous Alp4 with luciferase from the sea pansy Renilla reniformis (or R.luciferase) (Matthews et al., 1977). Alp4 was chosen based on the criteria that it is a low-expressing protein and is constantly but not periodically expressed during the cell cycle (Marguerat et al., 2012; Rustici et al., 2004).
By using the dual-reporter assay system (Promega), we measured and compared the levels of GBP–mCherry–F.luc fusion proteins that were expressed under different adh1 and nmt1 promoter variants (Fig. 3B). For nmt1 promoter (Pnmt1, Pnmt41, or Pnmt81)-driven GBP–mCherry-F.luc, we also measured yeast samples after the cultures were induced for 20 h in EMM2 medium in the absence of thiamine (Fig. 3B). As shown in Fig. 3B, all these different adh1 and nmt1 promoter variants provided a wide range of expression levels of tagged proteins. Consistent with previous studies (Basi et al., 1993; Forsburg, 1993), the full-length nmt1 promoter provided the strongest induction under induced conditions. However, the absolute induction ratios and relative luciferase activity between Pnmt1, Pnmt41, Pnmt81 and Padh1 were different from those reported previously. This is most likely caused by different reporter systems used in those and our studies.
Nevertheless, the quantitative data presented here should give a rough guide for expression levels from all these adh1 and nmt1 promoter variants, and be able to assist in deciding which promoter should be picked for specific tagging and forced-localization purposes. It would also help to choose one promoter to drive expression of one certain protein to a level comparable to its own promoter, namely mimicking the corresponding endogenous promoter strength. Apparently, it is more practical to take a trial-and-error approach towards promoters with different strengths when one needs to decide which promoter should be used for the final experiment, thus these promoters should be used on a case-by-case basis.
Application example I
Construction of integration strains expressing Padh-Dma1–GBP–mCherry and Pnmt-GBP–mCherry–Dma1 and rewiring of Dma1 to different subcellular loci
To validate our newly developed vectors for tagging proteins of interest with GBP or GBP–mCherry, and to test the efficiency of manipulating the subcellular localization of these fusion proteins, we constructed a fission yeast strain set carrying GBP–mCherry-tagged Dma1 as examples of integrant strains. Dma1 is a checkpoint protein, which couples mitotic progression with cytokinesis and is important in delaying mitotic exit and cytokinesis when kinetochores are not properly attached to the mitotic spindle (Guertin et al., 2002; Murone and Simanis, 1996). We first tagged Dma1 with C-terminal GBP–mCherry at its endogenous locus, with its expression driven by its native promoter (i.e. Pdma1-Dma1–GBP–mCherry, or in short Dma1–GBP–mCherry). Similar to Dma1–GFP (Guertin et al., 2002) and Dma1–mCherry (Fig. 4A), Dma1–GBP–mCherry also localized to SPBs and the division site (Fig. 4B). In order to compare protein levels of GBP–mCherry fusion proteins resulting from the different promoters and to examine whether the N- or C-terminal tagging leads to different cellular localization of Dma1, we also constructed strains with Dma1–GBP–mCherry driven by the adh1 promoter variants (Padh-Dma1–GBP–mCherry) and integrated at lys1+, and GBP–mCherry–Dma1 driven by the Pnmt variants (Pnmt-GBP–mCherry–Dma1) and integrated at leu1+ by performing one-step transformation of linearized plasmid DNA fragments (see Materials and Methods). Interestingly, after several attempts, we could successfully obtain strains carrying Pnmt1, Pnmt41 or Pnmt81, or Padh11, Padh21 or Padh81 promoters; however, we could not obtain a strain expressing Padh1-Dma1–GBP–mCherry, which uses the strongest constitutive promoter (data not shown), presumably because constantly overexpressed Dma1 leads to cytokinesis failure and lethality (Fig. S1), and this phenotype has been previously observed in cells overexpressing Dma1 under Pnmt1 (Guertin et al., 2002).
We observed that at least for Dma1, expression of the N- or C-terminally tagged protein achieved a similar localization pattern at SPBs or cell division sites to that of the endogenous protein, although the signal intensity of the GBP–mCherry fusion proteins varied according to the strength of each promoter (Fig. 4B). It is noteworthy that the signal intensities of the GBP–mCherry fusion proteins at SPBs were not always proportional to those at cell division sites. For example, although Padh11-Dma1–GBP–mCherry and Pnmt41-GBP–mCherry–Dma1 showed bright signals at cell division sites, their localization at SPBs was relatively weak (Fig. 4B). This is probably due to the different anchoring capability of Dma1 at these two sites. Interestingly, Pnmt1-GBP–mCherry–Dma1 did not show specific localization at SPBs or cell division sites, instead it accumulated in the cytoplasm and caused a multi-nucleate phenotype, which is indicative of cytokinesis failure (Fig. 4B). This phenotype is consistent with previous findings regarding overexpression of untagged Dma1 driven by Pnmt1 (Guertin et al., 2002). We also compared the protein levels of GBP–mCherry fusions of Dma1 by western blotting; we found that the level of Padh81-Dma1–GBP–mCherry was close to that of endogenous Dma1–GBP–mCherry under its own promoter, and Pnmt1-GBP–mCherry–Dma1 was most abundant after induction (Fig. 4C).
Next, we tested whether our Dma1–GBP–mCherry or GBP–mCherry–Dma1 constructs could be targeted to GFP-fused proteins localized at different subcellular compartments or structures through GBP–GFP binding. We chose a few GFP-fused proteins with specific localizations as ‘baits’, representing some major subcellular compartments or structures inside S. pombe cells, such as nuclear membrane (Amo1–GFP) (Pardo and Nurse, 2005), nucleoplasm (SV40-NLS–GFP–β-Gal) (Pasion and Forsburg, 1999), nucleolus (Gar2–GFP) (Gulli et al., 1995), SPB (Sid4–GFP) (Chang and Gould, 2000), cell ends (Tea1–GFP) (Mata and Nurse, 1997) and actomyosin ring (Cdc15–GFP) (Carnahan and Gould, 2003). We introduced these GFP-tagged markers into the Padh21-dma1-GBP–mCherry strain background by genetic crossing. Strikingly, almost all GFP-fused proteins tested could successfully anchor Dma1–GBP–mCherry to the sites where these ‘baits’ localized and bring about colocalization with Dma1 (Fig. 5A). One of the exceptions was Amo1–GFP, which only weakly recruited Dma1–GBP–mCherry to nuclear membrane and left the Dma1 localization at SPB and cell division sites almost unchanged (Fig. 5A). Very interestingly, the combination of Dma1–GBP–mCherry and Sid4–GFP was lethal, even when Dma1–GBP–mCherry was expressed at very low levels under Padh81 (Fig. 5B). This was most likely due to enhanced inhibitory ubiquitylation of Sid4 specifically and locally at SPBs by Dma1 (Johnson and Gould, 2011), and subsequent total failure of septation-initiation network (SIN) signaling and cytokinesis.
Our above proof-of-principle experiments showed how this localization manipulation approach proves of great use in the interrogation of the SPB-specific functions of Dma1. Overall, these experiments thus established a targeting system in which GBP- or GBP–mCherry-tagged proteins could be targeted to certain loci in the cell by their specific and strong binding to GFP-fusion proteins.
In principle, GBP- and GFP-tagged proteins should recruit their partners to respective subcellular sites to similar extents. However, very interestingly, so far, at least in fission yeast, GBP-tagged proteins have been almost always ‘drawn’ to GFP-fusion proteins but not the reverse direction, both in our study and published reports (Deng and Moseley, 2013; Dodgson et al., 2013; Grallert et al., 2013). Whether this is due to the strong localization capacity of the tested GFP-fusion proteins or some other unrecognized reasons awaits further investigations on more protein examples.
Application example II
Permanent and symmetric localization of Cdc7 at SPBs causes strong premature cell septation
Cytokinesis is the terminal step in cell division during which a single mother cell is physically divided into two daughters. In fission yeast, a signaling cascade termed the SIN plays a key role in the regulation of cytokinesis (Krapp and Simanis, 2008; McCollum and Gould, 2001). SIN involves three protein kinases (Cdc7, Sid1 and Sid2); a small GTPase (Spg1); a bipartite GTPase-activating protein (GAP) complex comprising Cdc16 and Byr4, which binds to and inhibits Spg1; a pair of SPB-resident proteins, Cdc11 and Sid4; and binding partners of Sid1 and Sid2, termed Cdc14 and Mob1, respectively (reviewed in Krapp and Simanis, 2008). Of these proteins, Spg1, Sid2 and Mob1 are detected at the SPB throughout the cell cycle, whereas Sid1, Cdc7 and Cdc14 localize asymmetrically to only one of the two SPBs (the newly generated SPB) during late mitosis and do not localize to the SPB in interphase, while conversely Byr4p and Cdc16p localize to the SPBs throughout interphase and then specifically to the ‘old’ SPB during late mitosis (reviewed in Krapp and Simanis, 2008; McCollum and Gould, 2001).
Based on the observations that inactivation of Cdc16, using the cdc16-116 mutation, or overexpression of Spg1 results in localization of Cdc7 to both SPBs symmetrically in late mitosis correlates with repeated rounds of septum formation without cell cleavage and thus a multi-septa phenotype (Guertin et al., 2000; Sohrmann et al., 1998), it has been concluded that asymmetric distribution of Cdc7 and SIN activity is crucially important for the SIN to trigger septation precisely (Guertin et al., 2000; Sohrmann et al., 1998). To develop an alternative way of promoting symmetric localization of Cdc7 to both SPBs during mitosis, we tried to utilize Sid4–GFP as the ‘bait’ to recruit Cdc7–GBP–mCherry permanently to SPBs. We chose Sid4 as the targeting protein due to the fact that as one of the scaffold proteins, Sid4 is a constitutive resident protein at SPB throughout the whole cell cycle (Krapp et al., 2001; Morrell et al., 2004). We first tried to construct strains with Padh-Cdc7–GBP–mCherry variants integrated at lys1+ and Pnmt-Cdc7-GBP–mCherry variants integrated at leu1+ (Fig. 6). Similar to the case for Padh1-Dma1–GBP–mCherry, we could not obtain the strain expressing Padh1-Cdc7–GBP–mCherry, which used the strongest constitutive promoter Padh1 (data not shown), most likely due to the intolerance of fission yeast cells to the permanently overexpressed Cdc7 (Fig. S2). We then introduced Sid4–GFP into the obtained viable strains carrying Cdc7–GBP–mCherry. Interestingly, all strains with integrated Padh-Cdc7–GBP–mCherry variants were lethal in the Sid4–GFP background (Fig. 7A), and Pnmt1-Cdc7–GBP–mCherry also became inviable, even without induction when Sid4–GFP was present (Fig. 7A), suggesting that, possibly, its forced loading at SPB after leaky expression of Pnmt1-Cdc7–GBP–mCherry when thiamine is present was enough to drive multiple septa formation and lethality.
We then compared the frequency of septation in strains that simultaneously carried Pnmt81- or Pnmt41-driven Cdc7–GBP–mCherry and Sid4–GFP, before and after induction of the expression of tagged Cdc7 for various time periods. We found that the presence of Sid4–GFP always increased the frequency of multi-septa cells and, strikingly, expression of Pnmt41-Cdc7–GBP–mCherry driven by the weaker nmt1 promoter variant together with Sid4–GFP could result in a similar effect of untimely septation to that of expression of only Pnmt1-Cdc7–GBP–mCherry – which is driven by the strongest nmt1 promoter – alone without Sid4–GFP (Fig. 7B,C). Even more remarkably, the high frequency of the multi-septa phenotype was easily induced in the Pnmt81-Cdc7–GBP–mCherry strain when Sid4–GFP was present (Fig. 7B,C). These effects certainly correlated with enhanced localization of Pnmt-Cdc7–GBP–mCherry at SPBs that was promoted by permanently localized Sid4–GFP (Fig. 7B). Therefore, combining Cdc7–GBP–mCherry and Sid4–GFP proved to be an elegant tool for the generation of conditional disruption of asymmetric distribution of Cdc7 during mitosis.
Tips for easy and efficient construction of integration vectors with GBP or GBP–mCherry tags
Practically, it is difficult to predict how much of the GBP or GBP–mCherry fusion proteins should be expressed in S. pombe cells in order to obtain the effect on manipulation of protein targeting as desired. Therefore, it may be necessary to try several promoters with distinct strengths. We compiled some technical tips during experimental procedures to facilitate the construction of pJK148- or pUC119-based GBP- or GBP–mCherry-tagging vectors carrying the gene of interest.
First, to construct pJK148- or pUC119-based tagging vectors carrying the series of variant nmt1 or adh1 promoters, you only need to first construct one pJK148- or pUC119-based tagging vector with one certain promoter, for example, Pnmt1 for a pJK148-based vector and Padh1 for a pUC119-based vector. Then, site-directed mutagenesis of the TATA-box sequence can be employed to easily change the promoter strength by using the procedures described (Mao et al., 2011) and the primers listed in Table S3.
Second, it is a good idea to design a few universal primers for amplifying vector backbones by PCR that can be used for cloning any gene of interest into pJK148- or pUC119-based tagging vectors by using T-type cloning procedures (Yang et al., 2013). This should greatly decrease the cost of primers, and the successfully amplified vector backbones can be stored and used for cloning different gene fragments.
Third, sometimes it might be required to bring two, or more than two, proteins to specific loci inside fission yeast cells, for example, two subunits that are essentially required for assembly of a fully functional protein complex. In this case, two or three encoded proteins can be tagged with GBP alone or GBP–mCherry fusion using the vectors constructed in this study at their endogenous genomic loci or integrated ectopically at either leu1+ or lys1+ loci without bringing conflict to integration-site selection. The efficiency of targeting of the protein of interest can always be examined by visualizing the localization of GBP–mCherry fusions first, although eventually fusing proteins with GBP alone should be sufficient.
Linking subcellular localization of one protein to its function at specific loci inside cells is essential for cell biologists to thoroughly characterize this protein. Compared to the AA system, the GBP-targeting strategy provides an alternative way of manipulating or rewiring localization of target proteins not only from the nucleus to cytoplasm, but also from their original sites to various specific subcellular spaces, although it has a major limitation that GBP cannot be temporally controlled and reversibly liberated after binding to GFP. In this study, we developed a comprehensive set of vectors for tagging proteins of interest with GBP or GBP–mCherry fusion. Our examination of the effects on targeting of Dma1 or Cdc7 to desired loci in yeast cells seems to support the idea that the GBP-based technique is easier to achieve and more efficient for manipulating localization of proteins than commonly used methods based on fusions to heterologous localization proteins or signal peptides. Thus, our vector set provides a flexible collection when people need to manipulate the localization of any protein. In principle, these vectors can also be modified and extended to other systems, as long as the backbones and the selection markers are properly replaced.
MATERIALS AND METHODS
Preparation of bacterial strains, media and nucleic acids
Escherichia coli host strain DH5α was used to propagate plasmids in standard Luria Bertani (LB) medium. Standard molecular biology methods were used. Enzymes were used as recommended by the suppliers (New England Biolabs, Fermentas, Transgen Biotech and TIANGEN Biotech).
Fission yeast strains, media, chemicals and genetic methods
Schizosaccharomyces pombe strains used and created in this study are listed in Table S2. Liquid cultures or solid agar plates consisting of rich medium (YE5S) or synthetic minimal media (EMM2) with appropriate supplements were used as described previously (Moreno et al., 1991). Genetic crosses and general yeast techniques were performed as described previously (Moreno et al., 1991). EMM2 with 5 µg/ml of thiamine was used to repress expression from the nmt1 promoters. G418 disulphate and hygromycin B were purchased from Sigma-Aldrich and Sangon Biotech, respectively. These drugs were added in solid YE5S plates to generate final concentrations of 100 μg/ml where appropriate.
Construction of pFA6a-GBP-kanMX6 and pFA6a-GBP-mCherry-kanMX6
In order to construct pFA6a-based vectors carrying GBP or GBP–mCherry, a newly optimized ‘T-type’ enzyme-free cloning method (Yang et al., 2013) was employed. The coding sequences of GBP and GBP–mCherry were PCR amplified using two sets of primers with Pfu DNA polymerase (TIANGEN Biotech). One set of primers was non-tailed primers and the other set was tailed primers (#878-881, Table S3). The genomic DNA extracted from a strain carrying Tea1–GBP–mCherry (Dodgson et al., 2013) was used as template. A linear complementary backbone of pFA6a was also produced in two parallel PCRs with a high-fidelity DNA polymerase (KD plus; Transgen Biotech) using pFA6a-GFP-kanMX6 (Bahler et al., 1998) as template and two sets of non-tailed or tailed primers (#874-877, Table S3). The PCR products were gel-purified, mixed, heat-denatured and annealed as described previously (Chen et al., 2015). The annealed mixtures were then used to transform chemically competent E. coli cells. The resulting plasmids pFA6a-GBP-kanMX6 and pFA6a-GBP-mCherry-kanMX6 (Fig. 1A) were verified by double digestion with restriction endonucleases, and inserts were verified by sequencing. DNA sequences of these vectors and the representative vectors constructed below are available upon request.
Construction of pJK148-based vectors for C-terminal GBP or GBP–mCherry tagging
The coding sequences of GBP and GBP–mCherry were PCR amplified from pFA6a-GBP-kanMX6 and pFA6a-GBP-mCherry-kanMX6, respectively, to introduce a BamHI site at one end and a NcoI site at the other end. The resultant PCR fragments were digested with BamHI and NcoI, and ligated into the vector pREP41-EGFP(C), which had been digested using the same enzymes (Craven et al., 1998). Thus, two plasmids named pREP41-GBP(C) and pREP41-GBP-mCherry(C) were first constructed. Then, the GBP and GBP–mCherry fragments flanked by the nmt41 promoter (Pnmt41) and nmt1 terminator (Tnmt) sequences from the above plasmids were cloned into the pJK148 vector (Keeney and Boeke, 1994) using the ‘T-type’ enzyme-free cloning method with primers #677-684 (Table S3). This resulted in the construction of two final tagging vectors, pJK148-Pnmt41-GBP(C)-leu1+ and pJK148-Pnmt41-GBP-mCherry(C)-leu1+ (Fig. 1B). To obtain tagging vectors carrying different strength nmt1 promoters, the TATA box sequences in both vectors pJK148-Pnmt41-GBP(C)-leu1+ and pJK148-Pnmt41-GBP-mCherry(C)-leu1+ were replaced by sequences corresponding to Pnmt81 and Pnmt1 using Quikgene site-directed mutagenesis (Mao et al., 2011) with the following primers: #734 and #735, #602 and #603 (Table S3). This generated four vectors: pJK148-Pnmt81-GBP(C)-leu1+, pJK148-Pnmt1-GBP(C)-leu1+, pJK148-Pnmt81-GBP-mCherry(C)-leu1+ and pJK148-Pnmt1-GBP-mCherry(C)-leu1+ (Fig. 1B). DNA sequences of the multi-cloning sites of these vectors and the representative vectors constructed below are depicted in Fig. 2.
Construction of pJK148-based vectors for N-terminal GBP or GBP–mCherry tagging
To construct pJK148-based vectors for N-terminal GBP or GBP–mCherry tagging, the EGFP fragment was removed from pREP41-EGFP(C) (Craven et al., 1998) using Quikgene site-directed mutagenesis (Mao et al., 2011) and primer pair #608 and #609 (Table S3). This resulted in a plasmid pREP41-w/o-EGFP, retaining seven restriction cutting sites – NdeI, SalI, KpnI, BglII, XhoI, BamHI and NcoI. Then, the NdeI-BglII fragments carrying GBP or GBP–mCherry were PCR amplified from pFA6a-GBP-kanMX6 and pFA6a-GBP-mCherry-kanMX6, respectively, were cloned into pREP41-w/o-EGFP right after promoter Pnmt41. This generated two vectors, pREP41-GBP(N) and pREP41-GBP-mCherry(N). The GBP and GBP–mCherry fragments flanked by nmt41 promoter (Pnmt41) and nmt1 terminator (Tnmt) sequences from the above plasmids were cloned into the pJK148 vector using the ‘T-type’ enzyme-free cloning method, as with C-terminal tagging vectors pJK148-Pnmt41-GBP(C)-leu1+ and pJK148-Pnmt41-GBP-mCherry(C)-leu1+ described above. These procedures resulted in the construction of two N-terminal tagging vectors, pJK148-Pnmt41-GBP(N)-leu1+ and pJK148-Pnmt41-GBP-mCherry(N)-leu1+ (Fig. 1B). As for pJK148-based C-terminal tagging vectors, the TATA box sequences on both vectors were mutagenized in order to obtain four vectors with stronger or weaker nmt1-variant promoters: pJK148-Pnmt81-GBP(N)-leu1+, pJK148-Pnmt1-GBP(N)-leu1+, pJK148-Pnmt81-GBP-mCherry(N)-leu1+ and pJK148-Pnmt1-GBP-mCherry(N)-leu1+ (Fig. 1B).
Construction of pUC119-based vectors for C-terminal GBP or GBP–mCherry tagging
The original pUC119-based vector pHBKA81 (kindly provided by Yoshinori Watanabe, Tokyo University, Tokyo, Japan) carries the adh81 promoter (Padh81), adh1 terminator (Tadh1), hphMX6 drug resistance cassette, a 1.5 kb fragment of the truncated lys1+ coding sequence (lys1*), and five restriction cutting sites – NdeI, SalI, XbaI, BamHI and NotI – as its multi-cloning sites (Kawashima et al., 2007). To expand the choices of restriction cutting sites for cloning, a few more restriction cutting sites were introduced into pHBKA81 using Quikgene site-directed mutagenesis (Mao et al., 2011) and primer pair #492 and #493 (Table S3). This generated a plasmid that we named ‘pHBKA81-9 cutting sites’, which carries nine restriction sites – NdeI, XmaI, KpnI, XhoI, NheI, SalI, XbaI, BamHI and NotI – as its multi-cloning sites. Then, the BamHI–NotI fragments carrying GBP or GBP–mCherry were PCR amplified from pFA6a-GBP-kanMX6 and pFA6a-GBP-mCherry-kanMX6, respectively, and cloned into ‘pHBKA81-9 cutting sites’ after being digested by BamHI and NotI. This generated two vectors, pUC119-Padh81-GBP(C)-hphMX6-lys1* and pUC119-Padh81-GBP-mCherry(C)-hphMX6-lys1* (Fig. 1C). The TATA box sequences on both vectors were mutagenized in order to generate vectors with stronger adh1 promoter variants using Quikgene site-directed mutagenesis (Mao et al., 2011) with the following primer pairs: #515 and #516, #924 and #925, and #560 and #561 (Table S3). This generated six vectors for C-terminal GBP or GBP–mCherry tagging: pUC119-Padh21-GBP(C)-hphMX6-lys1*, pUC119-Padh21-GBP-mCherry(C)-hphMX6-lys1*, pUC119-Padh11-GBP(C)-hphMX6-lys1*, pUC119-Padh11-GBP-mCherry(C)-hphMX6-lys1*, pUC119-Padh1-GBP(C)-hphMX6-lys1* and pUC119-Padh1-GBP-mCherry(C)-hphMX6-lys1* (Fig. 1C).
Construction of pUC119-based vectors for N-terminal GBP or GBP–mCherry tagging
The NdeI–XhoI fragments carrying GBP or GBP–mCherry were PCR amplified from pFA6a-GBP-kanMX6 and pFA6a-GBP-mCherry-kanMX6, respectively, and cloned into ‘pHBKA81-9 cutting sites’ after being digested by NdeI and XhoI. This generated two vectors, pUC119-Padh81-GBP(N)-hphMX6-lys1* and pUC119-Padh81-GBP-mCherry(N)-hphMX6-lys1* (Fig. 1C), which can be used for N-terminal GBP or GBP–mCherry tagging. The vectors with stronger adh1 promoter variants (Padh21, Padh11 and Padh1) were generated in the same way using Quikgene site-directed mutagenesis (Mao et al., 2011) as pUC119-based vectors for C-terminal GBP or GBP–mCherry tagging. Finally, six more vectors for N-terminal GBP or GBP–mCherry tagging were generated: pUC119-Padh21-GBP(N)-hphMX6-lys1*, pUC119-Padh21-GBP-mCherry(N)-hphMX6-lys1*, pUC119-Padh11-GBP(N)-hphMX6-lys1*, pUC119-Padh11-GBP-mCherry(N)-hphMX6-lys1*, pUC119-Padh1-GBP(N)-hphMX6-lys1* and pUC119-Padh1-GBP-mCherry(N)-hphMX6-lys1* (Fig. 1C).
GBP or GBP–mCherry tagging and transformation of S. pombe cells
C-terminal GBP or GBP–mCherry tagging at endogenous loci of genes of interest was performed by PCR-based gene targeting using pFA6a-GBP-kanMX6 or pFA6a-GBP-mCherry-kanMX6 as templates (Bahler et al., 1998). To create the dma1-GBP-mCherry-kanMX6 strain, the same primer set was used as in a previous study (Guertin et al., 2002). For integration of C- or N-terminal GBP- or GBP–mCherry-tagged genes at either leu1-32 (for pJK148-based vectors) or lys1+ (for pUC119-based vectors) sites in the genome, the coding sequence of each gene of interest was first cloned into pJK148-based or pUC119-based tagging vectors (Fig. 1). Then, ∼1 µg of plasmid DNA was digested and linearized with NruI or Bsu36I (for pJK148-based vectors) or ApaI (for pUC119-based vectors) in a 20 µl reaction before transformation. Yeast transformation was performed using the lithium acetate method (Bahler et al., 1998; Keeney and Boeke, 1994). Transformants were selected on YE5S plates containing G418 (for tagging based on pFA6a-GBP-kanMX6 or pFA6a-GBP-mCherry-kanMX6 vectors) or hygromycin B (for pUC119-based vectors), or EMM2 plates lacking leucine (for pJK148-based vectors).
Correct tagging and integration was verified by using a colony PCR method. For confirmation of C-terminal GBP or GBP–mCherry tagging at endogenous loci of genes of interest, colony PCR was performed with a pair of oligonucleotide primers: gene of interest-specific forward primer and a universal reverse primer #281 (Table S3). For confirmation of proper integration at the lys1+ site of chromosome I, colony PCR was performed with a pair of primers: #530 and #531 (Table S3), which should result in a 1.8 kb PCR product; whereas for integration at the leu1+ site of chromosome II, primer pair (#194 and #736, Table S3) was used for amplification of a 2.2 kb fragment.
Construction of strains carrying GBP–mCherry–F.Luc and Alp4–R.luciferase
The coding sequence of firefly luciferase was PCR amplified using two sets of primers (#1305-1308, Table S3) and plasmid pFA6a-Firefly-luciferase-kanMX6 (Yu et al., 2013) as template. Linear complementary backbone of pJK148-Pnmt-GBP-mCherry(N) (where Pnmt is Pnmt81, Pnmt41 or Pnmt1) or pUC119-Padh-GBP-mCherry(N)-hphMX6-lys1* (where Padh is Padh81, Padh21, Padh11 or Padh1) was also produced in two parallel PCRs using two sets of non-tailed or tailed primers (#1206, #1207, #1210, #1211, and #1321-1324; Table S3). Then, by following the procedures of the ‘T-type’ enzyme-free cloning method as described previously (Chen et al., 2015; Yang et al., 2013), a series of GBP–mCherry–F.luc vectors was generated (Fig. 3A; Table S1); these vectors were linearized and integrated into yeast genome as described above.
In order to construct genomically tagged Alp4–R.luciferase, which can be used as an internal control for luciferase activity measurement, pFA6a-Renilla-luciferase-natMX6 (Yu et al., 2013) was used to add a C-terminal Renilla luciferase tag to the endogenous alp4+ locus to create the Alp4–R.luciferase (natR) allele using PCR-based gene targeting (Bahler et al., 1998).
Western blot analyses
Yeast cell extracts were prepared in lysis buffer (120 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 8 M urea, 0.6 M β-mercaptoethanol) using glass bead disruption and a FastPrep homogenizer (MP Biomedical), as described previously (Yu et al., 2013). For detection after PAGE electrophoresis, rabbit polyclonal anti-mCherry antibody (ab167453, Abcam) (Morozova et al., 2016) was used as the primary antibody (1:1000 dilution), and Cdc2 was detected using rabbit polyclonal anti-PSTAIRE (sc-53, Santa Cruz Biotechnology) (Yu et al., 2013) as a loading control (1:1000 dilution). Goat anti-rabbit-IgG conjugated to horseradish peroxidase (Pierce) was used as the secondary antibody at 1:10,000 dilution. Membranes were developed with ECL western blotting reagents (Pierce).
Luciferase activity assay in yeast cell lysate
Yeast strains carrying both GBP–mCherry–F.luc and Alp4–R.luciferase (natR) were used. Exponential-phase cultures were pre-grown in liquid yeast extract at 30°C (for strains carrying GBP-mCherry-F.luciferase-hphMX6-lys1* under the adh1-variant promoters) or induced in EMM2 without thiamine at 30°C for 20 h (for strains carrying GBP-mCherry-F.luciferase-leu1+ under the nmt1-variant promoters), and 1×107–10×107 cells were collected and washed once with sterile water. Firefly and Renilla luciferase activity in cell lysates was measured using the dual-reporter assay system (E1910, Promega), as described previously (Yu et al., 2013).
GFP–, mCherry– or GBP–mCherry-fusion proteins were observed in live cells. For DAPI (4′,6-diamidino-2-phenylindole) (cat. no. 10236276001, Roche) staining of nuclei, cells were fixed with 10% glutaraldehyde, washed in PBS and resuspended in PBS plus 1 μg/ml DAPI. Photomicrographs were obtained using a Nikon 80i fluorescence microscope coupled to a cooled CCD camera (Hamamatsu, ORCA-ER), and image processing and analysis was performed using Element software (Nikon) and Adobe Photoshop.
We are grateful to Dr Li-lin Du for bringing the high affinity between GBP and GFP variants to our attention. We thank Drs Paul Nurse (Cancer Research UK, London, UK), Kathy Gould (Vanderbilt University, Nashville, USA), Yoshinori Watanabe, Iain Hagan (Cancer Research UK Manchester Institute, Manchester, UK) and Fred Chang (Columbia University, New York, USA) for providing the plasmids or yeast strains, and Dr James Dodgson (University of Cambridge, Cambridge, UK) for providing the yeast strain carrying GBP–mCherry. We also thank Drs Li-lin Du and Chuan-hai Fu for critical reading of the manuscript and valuable comments.
This work was supported by the National Natural Science Foundation of China [grant numbers 31171298 and 31371360 to Q.W.J.], the National Natural Science Foundation of China for Fostering Talents in Basic Research (grant number J1310027) and by the Fundamental Research Funds for the Xiamen University (grant number 201410384072 to Y.H.C.).
The authors declare no competing or financial interests.