Multiple conserved domains of the nucleoporin Nup124p and its orthologs Nup1p and Nup153 are critical for nuclear import and activity of the fission yeast Tf1 retrotransposon

Abstract

The nucleoporin Nup124p is a host protein required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr in fission yeast. The human nucleoporin Nup153 and the Saccharomyces cerevisiae Nup1p were identified as orthologs of Nup124p. In this study, we show that all three nucleoporins share a large FG/FXFG-repeat domain and a C-terminal peptide sequence, GRKIxxxxxRRKx, that are absolutely essential for Tf1 retrotransposition. Though the FXFG domain was essential, the FXFG repeats themselves could be eliminated without loss of retrotransposon activity, suggesting the existence of a common element unrelated to FG/FXFG motifs. The Nup124p C-terminal peptide, GRKIAVPRSRRKR, was extremely sensitive to certain single amino acid changes within stretches of the basic residues. On the basis of our comparative study of Nup124p, Nup1p, and Nup153 domains, we have developed peptides that specifically knockdown retrotransposon activity by disengaging the Tf1-Gag from its host nuclear transport machinery without any harmful consequence to the host itself. Our results imply that those domains challenged a specific pathway affecting Tf1 transposition. Although full-length Nup1p or Nup153 does not complement Nup124p, the functionality of their conserved domains with reference to Tf1 activity suggests that these three proteins evolved from a common ancestor.

Figure 1.

FXFG repeats of Nup124p are not required for Tf1 activity. Multicopy plasmids containing full-length and mutant versions of Nup124 whose transcription was under the control of its native promoter or the low strength, nmt81 (no messsage in thiamine) promoter were transformed into a Δnup124 mutant strain YNB19, along with the Tf1 reporter plasmid pHL499-1. Genetic assays measured the ability of these mutant strains to reinstate levels of Tf1 cDNA recombination and transposition to that of epigenetically expressed WT levels. PROT^fs (YNB10) containing a Tf1-reporter plasmid with a protease frame shift that blocks the expression of (Tf1) protease, reverse transcriptase, and integrase, serves as a negative control for the assay. Because protease is involved in the processing of the Tf1 genome, all the activities of the retrotransposon are blocked as a result of the frame shift mutation. IN^fs (YNB11) contains a Tf1-reporter plasmid with a frame shift in the integrase so Tf1 transposition is decreased but not Tf1 cDNA recombination and serves as a negative control for the assay. S. pombe strains containing a Tf1-neo plasmid (pHL449-1) were grown as patches as shown, on EMM −ura dropout agar plates in the presence of thiamine (Tf1 Off) or in the absence of thiamine (Tf1 On). The transcription of the nmt1-Tf1-neo gene is induced in the absence of thiamine (Tf1 On). After 4 d of incubation at 32°C, these plates were replica printed to medium containing 5-FOA (100 mg/ml) to eliminate the URA3-Tf1-neo plasmid. Finally, 5-FOA^r patches were printed to medium containing both 5-FOA + G418 and incubated at 32°C for 38 h to detect strains that became resistant to G418 as the result of insertions of the neo-marked Tf1 element into the genome. For cDNA recombination assays, strains with the neoAI-marked Tf1 plasmid were induced for the expression of Tf1 on EMM −Ura plates without thiamine for 4 d at 32°C. The plates were then replica printed to YES medium that contained 500 μg of G418/ml. Recombination between cDNA and cellular transposon sequences was scored on the G418 plates after 38 h of growth at 32°C. The numbering of the patches in the figure corresponds to strains diagrammatically represented in the bottom panel. Each oval shape in the diagrammatic representation indicates one FXFG repeat. (1) Epigenetically expressed WT (3HA:nup124), YNB58; (2) Null mutant containing an empty vector (nmt81:empty vector), YNB893; (3) PROT^fs, YNB10; (4) IN^fs, YNB11; (5 and 6) Nup124ΔFXFG1-2, YNB59; (7 and 8) Nup124ΔFXFG3-7, YNB235; (9 and 10) Nup124ΔFXFG8-9, YNB236; (11 and 12) Nup124ΔFXFG10-11, YNB65; (13 and 14) Nup124ΔFXFG3-7*^, YNB 1086; (15 and 16) Nup124ΔFXFG3-7*^, FXFG1-2,8-11 AAAA, YNB1140; and (17 and 18) Nup124ΔFXFG, YNB1052 (see Table 2 for details).

Figure 2.

Sequence alignment of Nup124p, Nup1p, and Nup153 FXFG-repeat domains. The alignment of the C-terminal region of SpNup124p with the C-terminal domain of ScNup1p and HuNup153. Identical residues are boxed in black and similar/conserved residues in gray. Amino acid ranges are indicated at the start and finish of each sequence.

Figure 3.

FXFG/FG-domains of Nup1p and Nup153 are functional in Nup124p in the context of Tf1 activity. (A) Disorder amino acids (% by frequency) in the indicated FXFG/FG region of SpNup124p, ScNup1p, HuNup153, and SpNup120p (a random non-repeat region corresponding to the C-terminal half of the protein) (B) Yeast strains expressing full-length Nup124p (YNB 1064), Nup124p with FXFG deletion (YNB 1052), chimeras of Nup124p with the indicated FXFG repeat (YNB 1117, Nup124pΔFXFG:Nup1pFXFG, or YNB 1096, Nup124pΔFXFG:Nup153FXFG), or a non-repeat–bearing chimeric fusion protein (YNB 1201, Nup124pΔFXFG:Nup120p no-FXFG) were assayed for Tf1 activity as described earlier. +++, Tf1 activity equivalent to the epigenetically expressed WT nup124 (YNB 1064); −, lack of Tf1 activity comparable to the null mutant (see Table 2 for details).

Figure 4.

Identification of a highly conserved peptide sequence xRKIxxxxSRRKx at the C-terminus of SpNup124p, ScNup1p, and Nup153. (A) Sequence alignment of the C-terminal 13-amino acid region of Nup124p (AA 1147-1159), Nup1p, and Nup153 from various species. Representatives were chosen from a broad selection of eukaryotes: S. pombe (Sp), S. cerevisiae (Sc), Human (Hu), Mouse, Mus musculus (Mus), Rattus norvegicus (Rat), Xenopus laevis (Xe), and Gallus gallus (Gg). (B) Tf1 activity of wild-type and mutant Nup124p at the C-terminal polypeptide, GRKIAVPRSRRKR (Nup124pCT) was measured as described in Materials and Methods. (C) The amino acids 1151-1155 (AVPRS) of the C-terminal polypeptide were mutated or deleted. Their effect on Tf1 activity was measured. (D) Tf1 activity was measured upon swapping the C-terminal peptide of Nup1p and Nup153 with the C-terminal peptide of Nup124p (YNB1058 and YNB1054, respectively), and swapping followed by addition of an arginine residue at the C-terminus (YNB1062 and YNB1215). Tf1 activity was also assayed in the presence of Nup1p (YNB 1017), upon addition of an arginine to Nup1p (YNB1098), Nup153 (YNB967) and upon swapping the C-terminal 12 amino acids of Nup153 with the C-terminal 13 amino acids of Nup124 (YNB1217; see Table 2 for details).

Figure 5.

Mutations in Nup124p prevent Tf1-Gag from being imported into the nucleus resulting in loss of Tf1-transposon activity. YNB19 was transformed with Tf1-Gag:YFP and 3HA-tagged Nup124p (YNB549). Mutant Nup124p strains are shown, and their details provided in Table 2. Cells were grown in −Ura-Leu medium with or without 15 μM thiamine. Cells were processed and visualized as indicated in Materials and Methods. Only cells grown in the absence of thiamine are shown. Strains shown were also tested for their ability to support Tf1 activity. +++, Tf1 activity equivalent to the epigenetically expressed WT (YNB549); −, lack of activity.

Figure 6.

Overexpression of the FXFG domain (Nup124pFXFG) or GRKIAVPRSRRKR (Nup124pCT) peptide sequence of Nup124p inhibits Tf1 activity without significantly affecting growth. (A) Genetic assays were conducted exactly as described in Figure 1 so as to measure the ability of epigenetically expressed mutants (patches 5-12) to reinstate levels of Tf1 activity in YNB16 (wild type). The indicated strains were grown in EMM −Ura-Leu+B1 (Tf1-off) and EMM −Ura-Leu-B1 (Tf1-on), and growth was measured as a function of optical density (OD 600 nm). Details of the indicated constructs provided in Table 2. (B) Multicopy plasmids containing fragments encoding the indicated amino acids, GRKIAVPRSRRKR from Nup124p (Nup124pCT) and a random sequence, YAHSDATMVCMFS, whose transcription was under the control of the high strength, nmt1 promoter were tested in a WT strain (YNB16) for their effect on Tf1 activity. (1) Null mutant containing an empty vector served as a negative control, (YNB893); (2-5) nmt1:GFP, YNB1253; (6-9) nmt1:GFP-GRKIAVPRSRRKR, YNB1066; (10-17) nmt1:GFP-YAHSDATMVCMFS, YNB1219; (18) PROT^fs(YNB10) described earlier (19) IN^fs (YNB11) described earlier. The indicated strains were grown in EMM −Ura-Leu+B1 (Tf1-off) and EMM −Ura-Leu-B1 (Tf1-on), and growth was measured as a function of optical density (OD 600 nm; see Table 2 for details).

Figure 7.

Tf1-Gag is mislocalized upon over expression of the Nup124p, Nup1p, and Nup153-FXFG/FG domains in the WT strain. YNB16 was transformed with RFP:Nup124pFXFG (YNB1223) and RFP:Nup120p seq (YNB1221) along with nmt1:Tf1-Gag:YFP. YNB 16 was also transformed with RFP:Nup124pFXFG (YNB1232) and RFP:Nup120p seq (YNB1230) alone (see Table 2 for details). Cells were grown in EMM −Ura-Leu medium with or without 15 mM thiamine. Only cells grown in the absence of thiamine are shown.

Figure 8.

Tf1-Gag is mislocalized upon over expression of the C-terminal GRKIAVPRSRRKR (Nup124pCT) polypeptide in a WT strain. YNB16 was transformed with nmt1:RFP-GRKIAVPRSRRKR (YNB1198) or nmt1:RFP-YAHSDATMVCMFS (YNB1191) along with nmt1:Tf1-Gag:YFP. YNB16 was transformed with nmt1:RFP-GRKIAVPRSRRKR (YNB1197), nmt1:RFP-YAHSDATMVCMFS (YNB1189) and nmt1:Tf1-Gag:YFP (YNB1234) alone. Cells were grown in EMM −Ura-Leu medium with or without 15 mM thiamine. Only cells that were induced are shown (see Table 2 for details).

Figure 9.

Coimmunoprecipitation of Nup124p with Tf1-Gag, Kap95, Imp1p, and Cut15p from rabbit reticulocyte lysates. (A) The following sequences: Nup124, Tf1-Gag, Kap95, Imp1, Cut15, and Grn1:GFP from BNB401, BNB706, BNB659, BNB704, BNB702, and BNB373, respectively (Supplementary Table S1), were transcribed and translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine. Translated products were mixed in various combinations as indicated. Coimmunoprecipitation assays were performed as described in Materials and Methods with the appropriate antibodies, as indicated. (B) Nup124AA1-570 (BNB566) and Nup124 AA571-1159 (BNB761) were transcribed and translated using the rabbit reticulocyte lysate in the presence of [³⁵S]methionine. The products were incubated with Tf1-Gag (BNB706) and immunoprecipitated with antibodies as indicated. (C) Grn1p (BNB338), a nucleolar protein of S. pombe was incubated with Nup124 (BNB401) and Tf1-Gag (BNB706) and immunoprecipitated with antibodies as indicated in Materials and Methods. The immunoprecipitates were resolved on 4-15% SDS-PAGE and detected by fluorography. The relative mobilities of full-length/+tag proteins are indicated by arrows.