NUP82 and NUP84 have accumulated elevated levels of non-synonymous substitutions

We first set out to determine which nuclear pore complex-related genes might be important in the evolved control of Tys. Obviously, genes that have remained unchanged over the speciation of Saccharomyces yeast would be unlikely to fall into this class. Instead, as a screening tool we sought genes that have diverged significantly in sequence from one yeast species to the next. We are particularly interested in genes with evidence for natural selection underlying these sequence changes, rather than genes that have diverged in sequence simply by the forces of random genetic drift. Natural selection can be detected in genes as follows. Typically, selection operates on non-synonymous substitutions (changing the encoded amino acid) more significantly than on non-synonymous mutations (silent, not changing the encoded amino acids). Gene regions that have experienced repeated rounds of natural selection in favor of protein-altering mutation therefore exhibit a characteristic inflation of the rate of non-synonymous (dN) DNA substitutions compared to synonymous (dS) substitutions (denoted by dN/dS > 1) [61]. Because non-synonymous mutations occur more often than synonymous mutations by random chance, computational models have been developed that use statistical frameworks to account for these unequal substitution rates [62–64]. The mode of evolution that we are seeking (dN/dS > 1) is considered to be somewhat rare in eukaryotic genes. Instead, most genes experience purifying selection (dN/dS < 1), where protein sequence is conserved over evolutionary time due to the important and complex roles that most proteins play in cellular homeostasis.

We examined the evolution of 29 yeast genes encoding nucleoporins and 22 genes encoding karyopherins for evidence of codons with dN/dS > 1. For each gene, we gathered nucleotide sequences from six divergent Saccharomyces species (S. cerevisiae, S. paradoxus, S. mikatae, S. kudriavzevii, S. arboricolus and S. bayanus) [65–67]. Next, we constructed DNA alignments of the various genes and fit these to two different models of codon evolution using the Phylogenetic Analysis by Maximum Likelihood (PAML) package [64]. Evolutionary model M7 was used as our null model and assumes that all codons within a gene are evolving conservatively (dN/dS > 1 not allowed), whereas model M8 allows for some codons to exhibit an elevated evolutionary rate (dN/dS ≥ 1). Model M7 was rejected in favor of M8 (p<0.05) for four nucleoporin genes: NUP84, NUP1, NUP116 and NUP82 (Fig 2B). The null model was not rejected for any karyopherins (S1 Fig). Interestingly, one of these nucleoporin genes, NUP84, is also the only nuclear pore-related gene found in three different knockout screens as important for Ty mobility (Fig 2A). NUP133, NUP120, and NUP170, which were found in two independent genetic screens (Fig 2A) did not pass the threshold of significance (p>0.05; Fig 2B), and so were not investigated further. The remaining three nucleoporin genes under positive selection (NUP1, NUP116 and NUP82) are essential genes within S. cerevisiae (Fig 2B, bottom), and of these, only NUP116 has been directly tested and demonstrated to be involved with Ty replication [25].

Various statistical tests have been designed to detect positive selection, all of which take different approaches to modeling the rates of nonsynonymous and synonymous changes that have occurred in a given gene alignment [69]. We next evaluated NUP84, NUP1, NUP116, and NUP82 with additional tests for positive selection, FEL and REL [68]. We found that all four nucleoporin genes showed evidence of positive selection using at least one of these additional tests (Fig 2C). Furthermore, three of these genes (NUP1, NUP82, and NUP116) were previously identified as evolving rapidly in a whole genome evolutionary study of five Saccharomyces yeast species performed by Scannell et al. [67]. In contrast, NUP133 and four other nucleoporins with the least support for rejection of the M7 null model (NDC1, NSP1, NUP57 and SEH1; Fig 2B), passed zero or only one of these tests (Fig 2C). We next turned to functionally testing the biological relevance of the observed evolutionary signatures identified within nuclear pore complex-related genes.

A novel GFP reporter of Ty mobility

We first built a quantitative, GFP-based assay system for Ty mobility, which is a variation of a previous assay used in this field [70]. In this system, a plasmid-mounted Ty1 genome from Saccharomyces cerevisiae was encoded on the Watson (sense) strand, and was engineered to contain an internal GFP gene on the Crick (anti-sense relative to the transcript) strand of the DNA (Fig 3A). To prevent its expression directly from the plasmid vector, the GFP gene was engineered to contain an antisense intron (on the Watson strand). Thus, only after the full-length Ty1-GFP transcript has been spliced, reverse transcribed, and integrated into the S. cerevisiae genome can the GFP gene be expressed. GFP expression is regulated by the inducible copper-sensitive CUP1 promoter (Fig 3A). Experiments were performed with two different introns within the GFP gene in order to determine which was more efficiently spliced from the transcript produced. The more efficient splicing occurred using the S. cerevisiae ACT1 intron (ACT1i) (Fig 3B). GFP-positive cells were only detected by flow cytometry after galactose was added to the growth medium to initiate Ty1 transcription, and subsequent addition of CuSO₄ to induce expression of the GFP reporter (Fig 3C). We tested our Ty1 mobility reporter in isogenic strains deleted for five genes known to be important for efficient Ty1 mobility: BY4741 xrn1Δ, nup84Δ, nup133Δ, bud22Δ, and xrs2Δ [51,52,55]. Indeed, we see a significant decrease in Ty1 mobility in each deletion strain compared to the wild-type BY4741 background (Fig 3D). As a control, we show that a strain deleted for NUP100, which is important for Ty3 mobility [56], but not known to be important for Ty1, supports a level of mobility that is not significantly different from that of a wild-type strain (Fig 3D).

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Fig 3. A novel GFP-based reporter of Ty1 mobility.

(A) An overview of the GFP-tagged Ty1 plasmid. Ty1 transcription is induced by activation of the GAL1 promoter that produces a long Ty1 transcript including an internal GFP gene and an ACT1 intron (ACT1i). The spliced transcript has the ACT1i removed, which then provides a template for Ty1 protein production and reverse transcription. Ty1 cDNA is imported into the nucleus and integrated into the S. cerevisiae genome. The GFP gene is then induced from the CUP1 promoter by CuSO₄ to report successful integration events. (B) RT-PCR was used to assess splicing of RNA with ACT1i versus an artificial intron (AI) within the GFP gene (primer positions marked by red arrows). Spliced RNA transcripts (Sp) were mainly detected upon induction of the transcription by the GAL1 promoter using galactose (Gal). Growth on dextrose (Dex) inhibits the GAL1 promoter and the production of RNA transcripts. Plasmid DNA was used as a positive control to allow the PCR amplification across intron-containing GFP. “Un Sp” indicated the detection of unspliced RNAs. (C) Flow cytometry analysis shows that GFP is only expressed under conditions of galactose induction of Ty1 expression followed by CuSO₄ induction of GFP. (D) The effect of six different gene deletions on Ty1 mobility, relative to wild-type S. cerevisiae. The relative mobility was measured as a percent of GFP positive cells after induction of the Ty1-GFP reporter, and was repeated independently, three times (error bars: standard error, n>3; **Tukey–Kramer method, p<0.05). All values are normalized to wildtype.

https://doi.org/10.1371/journal.pgen.1007325.g003

NUP84 evolution modulates Ty1 mobility within S. cerevisiae

NUP84 is under positive selection and disruption of the gene affects both Ty1 and Ty3 replication (Fig 2). We wished to test whether the evolution of NUP84 over yeast speciation has altered interactions with Tys. To test this, we replaced NUP84 within the S. cerevisiae genome (NUP84^S.cer) with NUP84 from diverse Saccharomyces species (S. mikatae, S. kudriavzevii and S. bayanus) as outlined in Fig 4A. These sequences encode Nup84p that are between 88% (S. mikatae) and 85% (S. bayanus) identical to the S. cerevisiae protein. As an isogenic control, we re-complemented the nup84Δ strain with S. cerevisiae NUP84. Chromosomal complementation of S. cerevisiae nup84Δ with each heterospecific (other species) NUP84 allele resulted in the restoration of normal growth and cellular morphology (Fig 4B and 4D), normal nuclear import (Fig 4C and 4D), and normal gene expression from the promoters used in our Ty1 GFP-based reporter (Fig 4E).

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Fig 4. The housekeeping functions of NUP84 are conserved across divergent Saccharomyces species.

(A) Top. A schematic representation of the NUP84 locus within S. cerevisiae engineered to either lack NUP84 (nup84Δ) or to express heterospecific NUP84 from S. cerevisiae (S. cer), S. mikatae (S. mik), S. kudriavzevii (S. kud) or S. bayanus (S. bay) along with the LEU2 selectable marker. Bottom. Successful genome engineering was confirmed by PCR amplification across the NUP84 locus to detect the replacement of KANMX4 with NUP84-LEU2 (primers marked as arrows). (B) The doubling time of NUP84-complemented strains in a liquid YPD medium compared to nup84Δ, and colony growth and morphology after 72 h of growth on a solid YPD medium. (C) General nuclear import function was assessed in the presence of heterospecific Nup84p or absence of Nup84p using a LexA-Gal4(AD) reporter protein with a SV40 nuclear localization signal (NLS) [71]. The LexA DNA binding domain and Gal4 activation domain (AD) initiate transcription of the β-galactosidase gene upon successful nuclear import. (D) Nuclear transport was also assessed by the steady-state localization of a GFP reporter protein containing a NLS from PHO4 [72] and its cellular accumulation relative to a DAPI-stained nucleus within NUP84 complemented S. cerevisiae. (E) The effect of NUP84 complementation or deletion on the ability of S. cerevisiae to express GFP from each of the promoters used in the Ty1 GFP-based reporter (GAL1 (top) or CUP1 (bottom) promoters), using mean fluorescent intensity (MFI) detected by flow cytometry (error bars: standard error, n>3).

https://doi.org/10.1371/journal.pgen.1007325.g004

The null strain, and each of the four strains expressing wildtype or heterospecific NUP84, were transformed with the Ty1 GFP reporter described above. Relative to nup84Δ, cells complemented with NUP84^S.cer increased Ty1 mobility approximately 5-fold (Fig 5A). There were highly significant differences in the levels of Ty mobility among strains encoding heterospecific NUP84 (one-way ANOVA, p = 8.2 x 10⁻⁸), and levels of Ty1 mobility were significantly different in strains containing NUP84^S.mik, NUP84^S.kud, and NUP84^S.bay when compared to NUP84^S.cer (Tukey–Kramer method, p<0.05) (Fig 5A). We found that replacement of NUP84^S.cer with NUP84^S.kud increased Ty1 mobility by 32%, whereas NUP84^S.mik and NUP84^S.bay both significantly decreased mobility by 21% and 35%, respectively. To verify the observed differences in control of Ty1 mobility, we used Southern blotting to detect Ty1 integrations in the 5’ UTR of the SUF16 locus, as previously described [73]. We used our GFP reporter assay to initiate Ty1 mobility, with Ty1 genomic integrations only detected after induction by galactose (Fig 5B). Similar to our GFP reporter assay, fewer integrations were detected within strains encoding NUP84^S.mik and NUP84^S.bay compared to NUP84^S.cer. NUP84^S.cer and NUP84^S.kud had comparable levels of genomic integrations (Fig 5B). To further ensure the generality of our findings, we also measured Ty1 mobility on a single-copy plasmid under both high and low expression conditions. We used the integration of a HIS3 gene as a marker of successful Ty1 mobilization by assaying the appearance of colonies able to grow on a histidine deficient medium [70]. Again, we were able to observe that heterospecific substitutions were able to alter Ty mobility, even with a 6–9 fold decrease in overall mobility from a single copy plasmid (S2 Fig). Results were broadly consistent between the three assays, with the exception of the heterospecific swap of S. kudriavzevii NUP84, which reduced Ty1 mobility in the low copy assay (S2 Fig).

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Fig 5. Evolutionary differences between NUP84 of different Saccharomyces species alter levels of Ty1 mobility.

(A) Relative mobility of Ty1 and Ty3 within nup84Δ or nup84Δ complemented with heterospecific NUP84 from different Saccharomyces species. Asterisks designate complemented strains that have significantly different levels of mobility compared to the strain encoding NUP84 from S. cerevisiae (Tukey–Kramer method, p<0.05) (error bars: standard error, n>3). (B) Southern blot analysis of Ty1 integration in two independent clones upstream of the SUF16 locus, which contains Ty1 integration hotspots in its promoter [73]. PCR products across the SUF16 locus were run on a gel and then probed with a radiolabeled DNA probe specific to GFP in order to detect integration events.

https://doi.org/10.1371/journal.pgen.1007325.g005

These data show that evolutionary differences between NUP84 of different Saccharomyces species modulate the efficiency of Ty1 mobility in a species-specific manner, even though all host functions are conserved. Pairing Ty1 from S. cerevisiae with NUP84 of other species apparently decouples a finely co-evolved relationship, altering levels of Ty1 mobility. To support this model, we also assayed the impact of NUP84 evolution on Ty3 replication. We used a galactose inducible Ty3 with a HIS3 reporter gene and assayed the appearance of colonies able to grow on a histidine deficient medium [70,74–76]. In contrast to Ty1, we found that nup84Δ resulted in increased Ty3 mobility, as was previously reported [56]. However, each of the heterospecific NUP84 genes returned transposition to the lower level with no significant difference in mobility among strains encoding heterospecific NUP84 (one way ANOVA, p = 0.90) (Fig 5A). Collectively, these data suggest that the co-evolutionary dynamics are specific to NUP84 and Ty1.

NUP82 evolution alters both Ty1 and Ty3 mobility

Our evolutionary analysis also identified the gene NUP82 as being the highest scoring nucleoporin in our evolutionary screen (Figs 2B and S1), however no role for NUP82 has been reported in Ty biology. This could be because NUP82 is an essential gene and would have eluded detection in genome-wide knockout screens. To investigate whether NUP82 is involved in Ty replication, a dominant negative approach was adopted. Full- or partial-length portions of NUP82 were expressed in cells that are otherwise wild type at the NUP82 locus. These Nup82p constructs included the mutations D204A, F290A, Y295A, L393A, I397A, L402A, L405A and F410A (Nup82p^DFY-LILLF) that inactivate interaction with other nucleoporins and decouple it from the nuclear pore complex [77] (Fig 6A). Nup82p^DFY-LILLF is non-functional as a nucleoporin, therefore we reasoned that it would compete with wild-type Nup82p and have an inhibitory effect on mobility if Ty interacts with Nup82p to transit the nuclear pore. Indeed, the expression of the C-terminal helical domain of Nup82p (residues 433–713) significantly reduced Ty1 mobility, with the N-terminal β-propeller domain (residues 1–458) being dispensable for this effect (Fig 6B). Expression of any of the dominant negative NUP82 genes did not noticeably affect the growth of S. cerevisiae (Fig 6C) or general nuclear import (Fig 6D) compared to expression of the control gene MET17, which suggests that these proteins are not toxic to S. cerevisiae and do not disrupt the nuclear pore complex. In summary, this serves as preliminary evidence of a previously uncharacterized role for NUP82 in Ty1 replication.

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Fig 6. The expression of dominant negative NUP82 and its impact on Ty1 mobility.

(A) Left. A linear domain diagram of Nup82p^DFY-LILLF and derived deletion mutants [Nup82p(433–713) and Nup82p^DFY-LILLF(1–458)]. Asterisks mark the mutations that decouple Nup82p from the nuclear pore complex. Right. Western blot analysis to detect the expression of FLAG-tagged Nup82p^DFY-LILLF and its derivatives, compared to the expression of a control protein (Met17p) in the wild-type background (*Met17p degradation products). The effect of Nup82p^DFY-LILLF expression on (B) Ty1 mobility, (C) doubling time in a liquid medium and (D) the nuclear import of the reporter protein LexA-MBP-Gal4(AD), relative to the expression of MET17 (error bars: standard error, n>3; **Tukey–Kramer method, p<0.05).

https://doi.org/10.1371/journal.pgen.1007325.g006

Next, in a similar approach to that taken with NUP84, S. cerevisiae was engineered to express NUP82 from different Saccharomyces species to ascertain the impact of NUP82 evolution on Ty mobility. Due to the essential nature of NUP82, we used a NUP82/nup82Δ heterozygous diploid strain from the “synthetic genetic array” collection [78] as our starting strain for the genomic replacement of NUP82^S.cer. A customized SceI restriction endonuclease method was used to improve the efficiency of homologous recombination-based gene replacement (see methods) (Figs 7A and S3). S. cerevisiae encoding heterospecific NUP82 have a normal colony morphology, growth rate (Fig 7B), and no difference in GAL1 and CUP1 promoter expression (S4 Fig), suggesting that the cells are the same in measurable host functions. We tested the effect of NUP82 evolution on Ty1 mobility using the GFP fluorescence assay and, in contrast to our studies of NUP84, found that Ty mobility levels were similar in strains expressing NUP82^S.mik and NUP82^S.bay and NUP82^S.cer, but were significantly higher for strains complemented with NUP82^S.kud (Fig 7C). Thus, although NUP82 may be important for Ty1 mobility (Fig 6), we find that Ty1 seems mostly insensitive to the evolutionary differences between NUP82 of different species. We next assayed the replication of a Ty3 retrotransposon in the engineered NUP82 heterospecific strains. S. cerevisiae expressing NUP82^S.mik resulted in a significant >3-fold increase in Ty3 mobility, relative to NUP82^S.cer (Tukey–Kramer method, p<0.05) (Fig 7C). These data show that the evolutionary differences within Saccharomyces NUP82 can impact both Ty1 and Ty3 mobility, but predominantly Ty3. Together, we show that NUP82 appears to play a previously uncharacterized role in Ty mobility, and that Ty1 and Ty3 are differentially susceptible to evolutionary changes within NUP82.

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Fig 7. The evolution of NUP82 and its effect on Ty1 and Ty3 mobility within S. cerevisiae.

(A) A schematic representation of S. cerevisiae engineered to express NUP82 from different Saccharomyces species. Genome engineering was confirmed by PCR amplification across the NUP82 locus. (B) The doubling time of NUP82-complemented strains in a liquid medium. Colony growth and morphology of the engineered strains was monitored for 72 h on a solid YPD medium (error bars: standard error, n>3). (C) Relative mobility of Ty1 and Ty3 within strains complemented with NUP82 from different Saccharomyces species. Asterisks mark significant differences in Ty mobility compared to the strain encoding NUP82 from S. cerevisiae (error bars: standard error, n>3; **Tukey–Kramer method, p<0.05).

https://doi.org/10.1371/journal.pgen.1007325.g007

Plasmid construction

The ACT1 intron (ACT1i) and an artificial intron (AI) [114] were amplified by PCR with included primer-encoded flanking homology to GFP. This PCR product was inserted directly after the ATG start codon at the 5’ end of GFP by the “yeast plasmid construction by homologous recombination” method (recombineering) [115]. GFP(AI) and GFP(ACT1i) were amplified by PCR and introduced into pAG423-GAL-ccdB using TOPO-TA and Gateway cloning strategies (Thermo Fisher) to create pPAR061 and pPAR063, respectively. GFP(ACT1i) was also placed under the control of the CUP1 inducible promoter (456 bp upstream of CUP1 were cloned directly from the genome of S. cerevisiae) using recombineering. pCUP1-GFP(ACT1i) was used to replace HIS3(AI) within pGTy1-HIS3(AI) to create pPAR078. pPAR101, pPAR104, pPAR145 and pPAR181 were constructed by using PCR to create DNA encoding FLAG-tagged Nup82p^DFY-LILLF(1–458), Nup82p^DFY-LILLF and Nup82p (433–713) from pNOP-GFP-Nup82p^DFY-LILLF [77]. MET17 was amplified directly from the genome of S. cerevisiae. All PCR fragments were subsequently cloned into pAG414-GPD-ccdB via pCR8 using TOPO-TA and Gateway cloning strategies (Thermo Fisher). To assay nuclear import using the strategy outlined by Marshall et al. we first subcloned the LexA-MBP-GAL4(AD) cassette from pJMB1076n [71] into the pAG413 plasmid backbone using recombineering, essentially changing the selective marker on the plasmid from LEU2 to HIS3. For gene knockout, all plasmids were constructed by recombineering using NUP82 and NUP84 amplified from various Saccharomyces species. These nucleoporin genes were placed upstream of a selectable marker (LEU2 or KANMX6) and the entire cassette flanked by 1000 bp of sequence encompassing the 5’ and 3’ untranslated regions of NUP82 or NUP84 from S. cerevisiae. pPAR240 was constructed by first amplifying a LexA operator sequence upstream of the β-galactosidase gene from S. cerevisiae L40. PCR products were designed to contain flanking homology to ADE2 from S. cerevisiae and these PCR products were used to disrupt the ADE2 gene within pRS422 to create pPAR240. The DNA sequences from all constructed plasmids can be found in S2 File. A list of all relevant plasmids can be found in S1 Table. A list of all relevant yeast strains can be found in S2 Table.

Evolutionary analyses

Gene sequences from six species of Saccharomyces yeasts were obtained from publically available online resources. Maximum likelihood analysis of dN/dS was performed using the codeml program in PAML 4.1. Multiple protein sequence alignments were created and were manually curated to remove ambiguities before processing with PAL2NAL to produce accurate DNA alignments [116]. DNA alignments were fit to two models: M7 (codons fit to a beta distribution of dN/dS values, with dN/dS > 1 disallowed) and M8 (similar to model 7, but with dN/dS > 1 allowed). One model of codon frequency (f61) and a seed value of 0.4 for dN/dS (ω) was used (S1 File). Likelihood ratio tests were performed to evaluate which model of evolution the data fit significantly better with positive selection and inferred if we can reject M7 in favor of M8 with a p<0.05. REL and FEL codon based models were also used to detect sites under positive selection as implemented by the HyPhy package using the best substitution models chosen by Akaike information criterion (AIC) using the phylogenetic tree (Newick format):

((((S. paradoxus, S. cerevisiae), S. mikatae), S. kudriavzevii), S. arboricolus, S. bayanus) (S1 File).

Strain construction

Standard methodologies for PCR-based gene knockout and replacement were used to create all NUP84 strains in BY4741 (YPAR0130-0133) [117]. Strains YPAR0135-0138 were engineered to encode a LexA operator sequence upstream of the β-galactosidase gene, and were constructed by the disruption of the genomic copy of ADE2 using a PCR cassette amplified from pPAR240. Clones selected for their ability to grow on a medium lacking uracil and inability to grown on a medium lacking adenine. NUP82 gene replacement utilized a SceI-based method to increase the efficiency of the integration of NUP82 and KANMX6 by generating DNA double-stranded breaks at the NUP82 locus in S. cerevisiae (personal communication, Dr. C.M. Yellman). Using a diploid heterozygous knockout strain of NUP82 [78], KANMX6 at the NUP82 locus was replaced with the URA3 gene from K. lactis flanked by SceI sites amplified by PCR from pCMY-IT3. Gene replacement was carried out by the concomitant expression of SceI from pGAL1-SCEH and the LiAc transformation of a PCR-derived cassette encoding NUP82-KANMX6. NUP82/nup82Δ::NUP82-KANMX6 clones were selected by their ability to grow in the presence of 400 μg mL^-1 G418 and their resistance to 5-FOA (0.1% w/v). Haploid clones were isolated from the engineered diploid strains using the SGA selection protocol as described previously [78]. The correct insertions were confirmed by PCR of genomic DNA of the NUP82 locus to create strains YPAR0139, YPAR0143, YPAR0141 and YPAR0142. A PCR cassette was used to disrupt HIS3 in YPAR0139, YPAR0143, YPAR0141 and YPAR0142, clones were selected for their ability to grow on a medium containing hygromycin and inability to grown on a medium lacking histidine to produce strains YPAR0143, YPAR0145, YPAR0147 and YPAR0149.

Splicing of the ACT1 intron and insertion of an artificial intron within the GFP gene

Plasmids pPAR063 and pPAR061 were used to produce GFP transcripts containing either the ACT1 intron (ACT1i) or an artificial intron (AI) [114], respectively, by induction from a galactose inducible promoter. Cultures were grown to mid-log phase in liquid culture with raffinose as the sole carbon source. At OD₆₀₀ of ~1, galactose or dextrose was added to a final concentration of 2% and the cultures grown at 30°C for 2 h. Total RNA was extracted from these cultures (~2 x 10⁷ cells) using the RNeasy RNA extraction kit (Qiagen). 5 μg of RNA was treated with 1 U of DNase I at 37°C for 10 min before heat inactivation at 75°C for 10 min. RNA samples were then subject to two-step RT-PCR using Superscript III with the GFP-specific primers:

5’-AAGCTGACCCTGAAGTTCATCTGC-3’ and

5’-CGTTGTGGCTGTTGTAGTTGTACTCC-3’.

Ty1 mobility assays

Yeast strains to be assayed for their ability to support Ty1 mobility were transformed with either pPAR078 (GFP flow cytometry method), pGTy1(HIS3(AI)) [73] or pBDG606 [51]. To detect Ty1 mobility, single colonies from S. cerevisiae transformed with each plasmid were isolated for each experiment. Each experiment was performed at least three times. Colonies were first grown for 24 h in 2 mL raffinose -uracil complete medium at 30°C with agitation. 1 x 10⁵ cells from the saturated cultures were used to inoculate 15 mL of -uracil complete medium with either 2% galactose or 2% raffinose with 0.02% galactose, followed by growth for 5 days at room temperature with agitation. For pGTy1(HIS3(AI)) or pBDG606: Cultures were serially diluted and plated onto a -uracil complete medium and a -uracil -histidine complete medium, both with dextrose as a carbon source. Colonies were counted after 2 days growth at 30°C and the percentage mobility was calculated. For pPAR078: Cultures were diluted and allowed to reach early log phase growth (OD₆₀₀ ~0.05) before the addition of CuSO₄ to a final concentration of 0.5 mM. Cultures were grown for 9 h at 30°C before assaying for the presence of live, GFP positive cells using a BD LSRII Fortessa flow cytometer (San Jose, CA) running FACSDiva software (v6.1.3). GFP excitation was observed with a blue, 488 nm laser, while GFP emission was collected using 530/30 nm band pass filter and 502 nm long pass filter. Propidium iodide (PI) excitation was observed with a yellow-green, 561 nm laser, while PI emission was collected using 660/20 nm band pass filter and a 635 nm long pass filter. 100,000 gated events were collected using a forward scatter vs side scatter dot plot, with forward scatter showing relative particle size and side scatter showing internal complexity. All subsequent plots were generated from this gated population. Live cells were gated by staining cell populations with PI (final concentration 0.1 μg mL^-1) and GFP positive populations were gated by comparison with GFP negative populations of cells. Analysis of flow cytometry data was performed using FlowJo version 9.7.6.

Ty3 mobility assay

For quantification of Ty3 mobility, yeast cells were transformed with pPS3858, a URA3 marked galactose inducible Ty3-HIS3 [70,74–76]. The HIS3 gene is located at the end of POL and is anti-sense to Ty3, except for an artificial intron which is sense. The sense intron prevents production of His3p until after the full-length Ty3 RNA is transcribed, spliced, reverse transcribed and integrated into the genome. Colony transformants were selected on a synthetic medium with 2% glucose (SD) complete with amino acids but lacking uracil. Single colonies were inoculated into 2 mL of synthetic raffinose (–uracil) and grown for 24 h. Cultures were then brought to 5 mL and grown for ~8 h, after which OD₆₀₀ was measured and cultures were diluted back to an OD₆₀₀ of ~0.02 in 4.5 mL and grown overnight. The following morning, 500 μL of 20% galactose (2% final) was added to induce Ty3 expression; after 8 h of induction cultures were pelleted, washed in SD medium, serially diluted, and plated on both SD plates lacking histidine for growth of transposed cells and also YPD plates to determine total live cell counts.

Nup82p^DFY-LILLF expression assays

Plasmids constitutively expressing either Nup82p^DFY-LILLF, Nup82p truncation mutants or Met17p were introduced into a strain containing a Ty1 mobility reporter plasmid. Mobility assays were carried out as previously described above, but with the use of a double-dropout complete medium to maintain both episomal vectors.

Western blotting of Nup82p^DFY-LILLF and truncation mutants

Yeast lysates were prepared from 5 mL of stationary phase culture grown for 16 h in a yeast complete medium -leucine -tryptophan. Cell pellets were washed with 1 mL of chilled 25 mM Tris-HCl (pH 7.0), 10 mM sodium azide before incubation at 100°C for 3 min. 50 μL of SDS sample loading buffer (100 mM Tris-HCl, 5% SDS, 10% glycerol, 0.1% bromophenol blue, 2% β-mercaptoethanol, pH 6.8) was added to the boiled pellet with 200 μL of acid-washed glass beads (0.5 mm). Samples were vortexed for 10 min to disrupt yeast cells before the addition of another 80 μL of SDS sample loading buffer. Glass beads were pelleted by centrifugation (1500 × g, 2 min). 30 μL of each sample was loaded directly onto a precast Tris-glycine 10% SDS-PAGE gel (Biorad). Flag-tagged NUP82 mutants were detected via Western blot using a 1:4000 dilution of a primary mouse monoclonal anti-flag (Syd Labs #M20008). Secondary detection was carried out using a 1:2000 dilution of a goat anti-mouse horseradish peroxidase conjugated antibody (Thermo #32430).

Nuclear import assays

A LexA-MBP-GAL4(AD) fusion protein with or without an SV40 nuclear localization signal [71] was used to measure the efficiency of nuclear import within S. cerevisiae L40 or BY4741. 5 mL of a glucose-supplemented synthetic complete medium lacking the appropriate amino acid and grown overnight at 30°C with agitation. Cells were collected by centrifugation at 4000 × g for 30 seconds and the cell pellets suspended in 750 μL of ice-cold ddH₂O. Washed cells were again collected by centrifugation (13,000 x g for 30 seconds), and soluble proteins extracted by Y-PER buffer as per manufacturer’s instructions (Thermo). The lysate was assayed for β-galactosidase activity as described previously [118].

Fluorescence microscopy

The steady-state import of GFP-NLS was monitored within BY4741 transformed with pEB0836 as described previously [72].

Detection of Ty1 genomic integrations by Southern blotting

The detection of the integration of Ty1 containing GFP by Southern blotting was performed as previously described [73], in the various NUP84-complemented or deletion strains of S. cerevisiae. Total DNA was extracted from cell cultures using phenol:chloroform and ethanol precipitation, after 5 days of induction, as described in the Ty1 mobility assay protocol above. Southern blotting was carried out after agarose gel electrophoresis, as described previously [119], using Hybond-XL membranes (GE healthcare).

Promoter activity assay

To assay the activity of the GAL1 promoter we expressed GFP under the control of the GAL1 promoter and monitored the increase in the mean fluorescent intensity (MFI) compared to uninduced control cells. Cells were grown overnight to saturation at 30°C (CM -uracil, 2% raffinose) before being used to seed a 10 mL culture that was grown to log phase (OD₆₀₀ 0.1–0.5). Each 10 mL log phase culture was divided into two 5 mL cultures, supplemented with either 2% galactose or dextrose (final concentration) and grown for 6 h. Cultures were assayed for GFP fluorescence by flow cytometry using the same instrumentation as described above. The activity of the CUP1 promoter was assayed by analyzing MFI data derived from Ty1-GFP mobility assays.